CCL28: A Promising Biomarker for Assessing Salivary Gland Functionality and Maintaining Healthy Oral Environments.

The oral cavity serves as the primary path through which substances from the outside world enter our body. Therefore, it functions as a critical component of host defense. Saliva is essential for maintaining a stable oral environment by catching harmful agents, including pathogens, allergens, and chemicals, in the air or food. CCL28, highly expressed in mucosal tissues, such as the colon and salivary glands, is a chemokine that attracts CCR10/CCR3 expressing cells. However, the role of CCL28 in salivary gland formation remains unclear. In this study, we investigated the salivary gland structure in CCL28-deficient mice. Histological analysis showed decreased staining intensity of Alcian blue, which detects acidic mucous, reduced expression of MUC2, and higher infiltration of gram-positive bacteria in the salivary glands of CCL28-deficient mice. In addition, CCL28-deficient mice contained ectopically MUC2-expressed cells in the ducts and reduced the expression of cytokeratin 18, a marker for ductal cells, within the submandibular glands, resulting in decreased duct numbers. Additionally, the submandibular glands of CCL28-deficient mice showed reduced expression of several stem cell markers. These results suggest that CCL28 regulates saliva production via proper differentiation of salivary gland stem cells and could be a valuable biomarker of salivary gland function.

Oral Microbiota: The Influences and Interactions of Saliva, IgA, and Dietary Factors in Health and Disease.

Recent advances in metagenomic analyses have made it easier to analyze microbiota. The microbiota, a symbiotic community of microorganisms including bacteria, archaea, fungi, and viruses within a specific environment in tissues such as the digestive tract and skin, has a complex relationship with the host. Recent studies have revealed that microbiota composition and balance particularly affect the health of the host and the onset of disease. Influences such as diet, food preferences, and sanitation play crucial roles in microbiota composition. The oral cavity is where the digestive tract directly communicates with the outside. Stable temperature and humidity provide optimal growth environments for many bacteria. However, the oral cavity is a unique environment that is susceptible to pH changes, salinity, food nutrients, and external pathogens. Recent studies have emphasized the importance of the oral microbiota, as changes in bacterial composition and balance could contribute to the development of systemic diseases. This review focuses on saliva, IgA, and fermented foods because they play critical roles in maintaining the oral bacterial environment by regulating its composition and balance. More attention should be paid to the oral microbiota and its regulatory factors in oral and systemic health.

The altered production and property of saliva induced by ingesting fermented food ingredients affect the oral microbiome composition in mice.

Oral functions are diverse and critical to human health. Therefore, insufficient secretion or poor quality of saliva, which is secreted into the oral cavity and plays various roles, could have a crucial influence on the oral microenvironment and be associated with systemic disease development. Here, we investigated the effects of food ingredients on saliva quantity and quality, including fermented ones. Through the in vitro submandibular glands' organ culture analyses, we found that "Yomo gyutto," fermented Japanese mugwort (Artemisia princeps), altered the expression of aquaporin-5, a water channel protein. We also found that Yomo gyutto increased saliva volume, along with the amount of α-amylase in mice, and caused changes in the oral microbiome composition of mice. These results suggested that by ingesting Yomo gyutto, we could directly and effectively manipulate the quantity and quality of saliva secreted from the salivary glands, potentially altering the oral microbiome composition for individual health.

CCR3 blockage elicits polyploidization associated with the signatures of epithelial-mesenchymal transition in carcinoma cell lines.

Malignant features such as the acquisition of metastatic ability, stemness of cells, and therapeutic resistance of cancer cells are associated with epithelial-mesenchymal transition (EMT) accompanied by changes in motility and morphology. Recent reports implicated that the formation of polyploid giant cancer cells (PGCCs) in human malignancy correlated with the EMT processes. Chemokines are often involved in the regulation of cancer cell migration into tissues, and various types of human cancers exhibit enhanced expression of chemokine receptors, which could augment intrinsic potentials such as invasive activity, proliferating ability, and survival capacity in cancer cells. Nevertheless, the contribution of CCR3 in malignant cancer cells is controversial because it is a well-known primal receptor for the migration of eosinophils, one of the cells of the innate immune system. Here, we explored the blockage of chemokine receptor CCR3 in carcinoma cell lines and found that inhibition of CCR3 induced the formation of polyploid giant cells and stabilization of ß-catenin via the PI3K/Akt/GSK-3ß signaling pathway, which are processes associated with EMT. As a result of CCR3 inhibition, converted cells acquired enhanced mobile and proliferation abilities. In summary, these data indicate that modulation of the CCR3/PI3K/Akt/GSK-3ß signaling pathway regulates polyploidization associated with the EMT processes.

CCR4 plays a pivotal role in Th17 cell recruitment and expansion in a mouse model of rheumatoid arthritis.

T helper 17 (Th17) cells express CC chemokine receptor 4 (CCR4) and secrete cytokines such as interleukin-17A (IL-17A) and granulocyte macrophage colony-stimulating factor (GM-CSF), while dendritic cells (DCs) produce CC chemokine ligand 22 (CCL22), a CCR4 ligand, upon stimulation with GM-CSF. Th17 cells are known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). CCL22 has also been shown to be up-regulated in the synovial tissues of RA patients. Here, we investigated the role of CCR4 in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1J mice efficiently developed CIA as shown by erythema, paw swelling, joint rigidity, and joint destruction. Th17 cells were increased in the arthritic joints and regional lymph nodes (LNs) of CIA mice. A fraction of Th17 cells were also shown to produce GM-CSF. On the other hand, we observed no significant increases of Th2 cells or Treg cells, the T cell subsets also known to express CCR4, in these tissues. We further observed clusters of CCR4-expressing memory Th17 cells and CCL22-producing DCs in the regional LNs of CIA mice, supporting the role of the CCR4-CCL22 axis in the expansion of Th17 cells in the regional LNs. Compound 22, a CCR4 inhibitor, ameliorated the disease severity with reduction of Th17 cells in the arthritic joints and regional LNs and Th17-DC clusters in the regional LNs. We further confirmed that CCR4-deficient mice in the C57BL/6J background were highly resistant to CIA induction compared with wild-type mice. Collectively, CCR4 contributes to the pathogenesis of CIA and may thus represent a new therapeutic target for RA.

CCR4 Involvement in the Expansion of T Helper Type 17 Cells in a Mouse Model of Psoriasis.

Psoriasis is a chronic skin disease associated with T helper (Th)17-mediated inflammation. Because CCR4 is a major chemokine receptor expressed on Th17 cells, we investigated the role of CCR4 in a modified imiquimod-induced psoriasis model that showed enhanced skin infiltration of Th17 cells. CCR4-deficient mice had less severe skin disease than wild-type mice. Th17 cells were decreased in the skin lesions and regional lymph nodes of CCR4-deficient mice. In the regional lymph nodes of wild-type mice, CD44+ memory Th17 cells expressing CCR4 were found to be clustered with dendritic cells expressing CCL22, a ligand for CCR4. Such dendritic cell‒Th17 cell clusters were significantly decreased in CCR4-deficient mice. Similar results were obtained using the IL-23‒induced psoriasis model. In vitro, compound 22, a CCR4 antagonist, significantly reduced the expansion of Th17 cells in the coculture of CD11c+ dendritic cells and CD4+ T cells separately prepared from the regional lymph nodes of wild-type mice with psoriasis. In vivo, compound 22 ameliorated the psoriasis-like skin disease in wild-type mice with significant decreases of Th17 cells in the regional lymph nodes and skin lesions. Collectively, CCR4 is likely to play a role in the pathogenesis of psoriasis through the expansion of Th17 cells.

Kinase activity-independent role of EphA2 in the regulation of M-phase progression.

Cell division is a tightly regulated, essential process for cell proliferation. Very recently, we reported that EphA2 is phosphorylated at Ser897, via the Cdk1/MEK/ERK/RSK pathway, during M phase and contributes to proper M-phase progression by maintaining cortical rigidity via the EphA2pSer897/ephexin4/RhoG pathway. Here, we show that EphA2 kinase activity is dispensable for M-phase progression. Although EphA2 knockdown delayed this progression, the delay was rescued by an EphA2 mutant expression with an Asp739 to Asn substitution, as well as by wild-type EphA2. Western blotting analysis confirmed that the Asp739Asn mutant lost its EphA2 kinase activity. Like wild-type EphA2, the Asp739Asn mutant was localized to the plasma membrane irrespective of cell cycle. While RhoG localization to the plasma membrane was decreased in EphA2 knockdown cells, it was rescued by re-expression of wild-type EphA2 but not via the mutant containing the Ser897 to Ala substitution. This confirmed our recent report that phosphorylation at Ser897 is responsible for RhoG localization to the plasma membrane. In agreement with the M-phase progression's rescue effect, the Asp739Asn mutant rescued RhoG localization in EphA2 knockdown cells. These results suggest that EphA2 regulates M-phase progression in a manner independent of its kinase activity.

Involvement of Stat3 phosphorylation in mild heat shock-induced thermotolerance.

Thermotolerance is a phenomenon in which cells become resistant to stress by prior exposure to heat shock, and its development is associated with the induction of heat shock proteins (Hsps), including Hsp70. We previously showed that the expression of Hsp70 is regulated by the cytokine signaling transcription factor Stat3, but the role of Stat3 in thermotolerance is not known. In this study, we examined the possible involvement of Stat3 in the acquisition of thermotolerance. We found that severe heat shock-induced morphological changes and decreases in cell viability, which were suppressed by exposure to non-lethal mild heat shock prior to severe heat shock. This thermotolerance development was accompanied by Stat3 phosphorylation and the induction of Hsps such as Hsp105, Hsp70, and Hsp27. Stat3 phosphorylation and Hsp induction were inhibited by AG490, an inhibitor of JAK tyrosine kinase. Consistent with this, we found that mild heat shock-induced thermotolerance was partially suppressed by AG490 or knockdown of Hsp105. We also found that the Stat3 inhibitor Stattic suppresses the acquisition of thermotolerance by inhibiting the mild heat shock-induced Stat3 phosphorylation and Hsp105 expression. These results suggest that the mild heat shock-dependent stimulation of the JAK-Stat signaling pathway contributes to the development of thermotolerance via the induction of Hsps including Hsp105. This signaling pathway may be a useful target for hyperthermia cancer therapy.

EphA2 phosphorylation at Ser897 by the Cdk1/MEK/ERK/RSK pathway regulates M-phase progression via maintenance of cortical rigidity.

Successful cell division is accomplished by the proper formation of the mitotic spindle. Here, we show that EphA2 knockdown causes mitotic errors, including a delay in M-phase progression, asymmetric spindle positioning, multipolar spindles, and cell blebs. It has been known that EphA2 is phosphorylated at Tyr588, which is triggered by the ligand binding, and at Ser897 downstream of growth factor signaling. Upon mitotic entry, EphA2 is phosphorylated at Ser897, accompanied by a reduction in Tyr588 phosphorylation. This EphA2 phosphorylation at Ser897 is inhibited by MEK/ERK and 90 kDa ribosomal S6 kinase (RSK) inhibitors and is induced by the introduction of active cyclin-dependent kinase 1 (Cdk1) and cyclin B1. EphA2 knockdown-induced M-phase delay and cell blebs are rescued by wild type EphA2 expression but not by Ser897Ala mutant. The Ras homolog gene family member G (RhoG) guanine nucleotide exchange factor Ephexin4 interacts with EphA2 in a Ser897 phosphorylation-dependent manner, and its knockdown delays M-phase progression and causes RhoG delocalization. RhoG knockdown delays M-phase progression, and EphA2 knockdown-induced M-phase delay is partially rescued by the constitutively active RhoG mutant. These results suggest that, in EphA2-expressing cells, EphA2 phosphorylation at Ser897 participates in proper M-phase progression downstream of the Cdk1/MEK/ERK/RSK pathway because of its role in maintaining cortical rigidity via Ephexin4 and RhoG and thereby regulating mitotic spindle formation.-Kaibori, Y. Saito, Y., Nakayama, Y. EphA2 phosphorylation at Ser897 by the Cdk1/MEK/ERK/RSK pathway regulates M-phase progression via maintenance of cortical rigidity.

Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression.

Cell division is the process by which replicated chromosomes are separated into two daughter cells. Although regulation of M phase has been extensively investigated, not all regulating factors have been identified. Over the course of our research, small molecules were screened to identify those that regulate M phase. In the present study, the vascular endothelial growth factor receptor (VEGFR) inhibitors A83-01, SU4312, and Ki8751 were examined to determine their effects on M phase. Treatment of HeLa S3 cells with these inhibitors suppressed cell proliferation in a concentration-dependent manner, and also suppressed Akt phosphorylation at Ser473, a marker of Akt activation. Interestingly, cleaved caspase-3 was detected in Adriamycin-treated cells but not in inhibitor-treated cells, suggesting that these inhibitors do not suppress cell proliferation by causing apoptosis. A cell cycle synchronization experiment showed that these inhibitors delayed M phase progression, whereas immunofluorescence staining and time-lapse imaging revealed that the M phase delay was accompanied by misalignment of chromosomes and rotation of the mitotic spindle. Treatment with the Mps1 inhibitor AZ3146 prevented the SU4312-induced M phase delay. In conclusion, the VEGFR inhibitors investigated here suppress cell proliferation by spindle assembly checkpoint-induced M phase delay, via misalignment of chromosomes and rotation of the mitotic spindle.

A novel immunofluorescence method to visualize microtubules in the antiparallel overlaps of microtubule-plus ends in the anaphase and telophase midzone.

Cell division, in which duplicated chromosomes are separated into two daughter cells, is the most dynamic event during cell proliferation. Chromosome movement is powered mainly by microtubules, which vary in morphology and are organized into characteristic structures according to mitotic progression. During the later stages of mitosis, antiparallel microtubules form the spindle midzone, and the irregular formation of the midzone often leads to failure of cytokinesis, giving rise to the unequal segregation of chromosomes. However, it is difficult to analyze the morphology of these microtubules because microtubules in the antiparallel overlaps of microtubule-plus ends in the midzone are embedded in highly electron-dense matrices, impeding the access of anti-tubulin antibodies to their epitopes during immunofluorescence staining. Here, we developed a novel method to visualize selectively antiparallel microtubule overlaps in the midzone. When cells are air-dried before fixation, aligned α-tubulin staining is observed and colocalized with PRC1 in the center of the midzone of anaphase and telophase cells, suggesting that antiparallel microtubule overlaps can be visualized by this method. In air-dried cells, mCherry-α-tubulin fluorescence and ß-tubulin staining show almost the same pattern as α-tubulin staining in the midzone, suggesting that the selective visualization of antiparallel microtubule overlaps in air-dried cells is not attributed to an alteration of the antigenicity of α-tubulin. Taxol treatment extends the microtubule filaments of the midzone in air-dried cells, and nocodazole treatment conversely decreases the number of microtubules, suggesting that unstable microtubules are depolymerized during the air-drying method. It is of note that the air-drying method enables the detection of the disruption of the midzone and premature midzone formation upon Aurora B and Plk1 inhibition, respectively. These results suggest that the air-drying method is suitable for visualizing microtubules in the antiparallel overlaps of microtubule-plus ends of the midzone and for detecting their effects on midzone formation.