Human odontoblasts express functional TRPV2 channels

Background: The transient receptor potential (TRP) ion channels play a critical role in sensory physiology, where they act as transducers of thermal, mechanical and chemical stimuli. We have previously shown the functional expression of several TRP channels by human odontoblast-like cells and proposed their significance in odontoblast sensory perception. Functional expression of the mechano-sensitiveTRPV2 channel by human odontoblasts would further support a role for TRP channels in odontoblast physiology. Objective: The objective of the current study was to determine the functional expression of TRPV2 by human odontoblasts. Methods: Human dental pulp cells were cultured in the presence of 2 mM β-glycerophoshate to induce an odontoblast phenotype. TRPV2 gene expression was determined by qPCR employing custom designed FAM TRPV2 specific primers and probes (Roche, UK) and the Light Cycler 480 Probes Master (Roche). TRPV2 protein expression was determined following SDS-PAGE and Western blotting of cell lysate preparations. Functional expression of TRPV2 was investigated by Ca2+ microfluorimetry. Results: qPCR data indicated robust expression of TRPV2 in odontoblast-like cells. Western blotting revealed a discrete immunoreactive protein band indicating expression of TRPV2 in cell lysates. In functional assays, the chemical agonist of TRPV2, cannabidiol, was shown to elicit [Ca2+]i transients, that were reduced to baseline in the presence of the TRPV2 antagonist Tranilast, suggesting channel functionality in odontoblast-like cells. Conclusion: These results provide the first evidence for the functional expression of TRPV2 in human odontoblast-like cells, providing further support for the role of TRP channels in odontoblast physiology.

Gingival fibroblasts respond to the TRPV1 agonist capsaicin

Background: The oral cavity is a frontline barrier which is often exposed to physical trauma and noxious substances, leading to pro-inflammatory responses designed to be protective in nature. The transient receptor potential (TRP) super family of ion channels is believed to play a critical role in sensory physiology, acting as transducers for thermal, mechanical and chemical stimuli. Our understanding of the role of TRP channel activation in gingival and periodontal inflammation is currently limited. Gingival fibroblasts are the most abundant structural cell in periodontal tissues and we hypothesised that they may have a role in the inflammatory response associated with TRP channel activation. Objectives: The present study was designed to determine whether the TRPV1 agonist capsaicin could elicit a pro-inflammatory response in gingival fibroblasts in vitro by up-regulation of interleukin-8 (IL-8) production. Methods: Gingival fibroblasts were derived by explant culture from surgical tissues following ethical approval. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum (FCS) in 5% CO2. Following treatment of gingival fibroblasts with capsaicin, IL-8 levels were measured by ELISA. The potential cytotoxicity of capsaicin was determined by the MTT assay. Results: In gingival fibroblasts treated with the TRPV1 agonist capsaicin (10µM), IL-8 production was significantly increased compared with untreated control cells. Capsaicin was shown not to be toxic to gingival fibroblasts at the concentrations studied. Conclusion: The identification of factors that modulate pro-inflammatory cytokine production is important for our understanding of gingival and periodontal inflammation. This study reports for the first time that gingival fibroblasts respond to the TRPV1 agonist capsaicin by increased production of IL-8. Activation of TRPV1 on gingival fibroblasts could therefore have an important role in initiating and sustaining the inflammatory response associated with periodontal diseases

Functional expression of TRPV4 in human periodontal ligament cells

Background: Periodontal ligament (PDL) cells are exposed to physical forces in vivo in response to mastication, parafunction, speech and orthodontic tooth movement. Although it has been shown that PDL cells perceive and respond directly to mechanical stimulation, the nature of the ion channels that mediate this mechanotransduction remain to be fully elucidated. The transient receptor potential (TRP) superfamily of ion channels is believed to play a critical role in sensory physiology, where they act as transducers for thermal, chemical and mechanical stimuli. Recent studies have shown that members of the vanilloid (TRPV) and ankyrin (TRPA) subfamilies encode mechanosensitive TRPs. The vanilloid family member TRPV4 is one such non selective calcium permeable cationic channel which has been shown to be activated by chemical ligands, hypotonicity, and mechanical stimuli. Objectives: The objective of the current study was to investigate functional expression of TRPV4 in cultured human PDL cells. Methods: Human PDL cells were grown in Dulbecco's Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100UI/ml penicillin and 100μg/ml streptomycin. Cells in passage 4-6 were used in all experiments. TRPV4 functional expression was determined using ratiometric calcium imaging. Cultured cells were loaded with intracellular Ca2+ probe fura-2 and cells were then stimulated with the TRPV4 agonists, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), GSK1016790A or hypotonic solution. The TRPV4 antagonist RN 1734 was used to block the corresponding agonist responses. Results: PDL fibroblasts responded to application of TRPV4 agonists and hypotonic stimuli by an increase in intracellular calcium which was attenuated in the presence of the TRPV4 antagonist. Conclusions: We have shown for the first time the functional expression of the mechanosensitive TRPV4 channel in human PDL cells. The molecular identity and mechanisms of activation of mechanosensitive TRP channels in PDL cells merit further investigation.

Reduced epithelial suppressor of cytokine signalling 1 in severe eosinophilic asthma

Severe asthma represents a major unmet clinical need. Eosinophilic inflammation persists in the airways of many patients with uncontrolled asthma, despite high-dose inhaled corticosteroid therapy. Suppressors of cytokine signalling (SOCS) are a family of molecules involved in the regulation of cytokine signalling via inhibition of the Janus kinase-signal transducers and activators of transcription pathway. We examined SOCS expression in the airways of asthma patients and investigated whether this is associated with persistent eosinophilia.

Healthy controls, mild/moderate asthmatics and severe asthmatics were studied. Whole genome expression profiling, quantitative PCR and immunohistochemical analysis were used to examine expression of SOCS1, SOCS2 and SOCS3 in bronchial biopsies. Bronchial epithelial cells were utilised to examine the role of SOCS1 in regulating interleukin (IL)-13 signalling in vitro.

SOCS1 gene expression was significantly lower in the airways of severe asthmatics compared with mild/moderate asthmatics, and was inversely associated with airway eosinophilia and other measures of T-helper type 2 (Th2) inflammation. Immunohistochemistry demonstrated SOCS1 was predominantly localised to the bronchial epithelium. SOCS1 overexpression inhibited IL-13-mediated chemokine ligand (CCL) 26 (eotaxin-3) mRNA expression in bronchial epithelial cells.

Severe asthma patients with persistent airway eosinophilia and Th2 inflammation have reduced airway epithelial SOCS1 expression. SOCS1 inhibits epithelial IL-13 signalling, supporting its key role in regulating Th2-driven eosinophilia in severe asthma.

Low-dose salinomycin induces anti-leukemic responses in AML and MLL

Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response.
Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.