Yuko Imai

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels

Phosphatidylinositol 4,5-bisphosphate has a direct role in regulating receptor-operated TRPC channel activation and inactivation.

A self‐limiting regulation of vasoconstrictor‐activated TRPC3/C6/C7 channels coupled to PI(4,5)P2‐diacylglycerol signalling

•  From brain to digestive tract, electro‐chemical signals are broadly utilized to control the activity of the organs; however, the formation of such signals is very varied in each cell and still unknown in many cells. •  In this study, we found a novel mechanism for forming an electrical signal, produced by channels of the transient receptor potential canonical (TRPC) family of channels, which allow the permeation of ions such as sodium and calcium and are opened by the actions of hormones such as adrenaline and noradrenaline. •  Such hormones can activate an enzyme (phospholipase C) by which PI(4,5)P2, a member of the membrane lipid ‘phosphoinositide’, is degraded: the degradation of PI(4,5)P2 to produce an agonist (diacylglycerol) involved in the opening of TRPC channels, while the degradation itself is surprisingly critical to the closing of these channels. •  As a result of such a self‐limiting effect via membrane lipid degradation, TRPC channels can produce a unique electro‐chemical signal which is tightly bound to the arrangement of membrane lipid and hormones. •  Differential sensitivity for PIP2 of TRPC

Counteracting effect of TRPC1-associated Ca2+ influx on TNF-α-induced COX-2-dependent prostaglandin E2 production in human colonic myofibroblasts.

TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E(2) (PGE(2)) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca(2+). In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca(2+)-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca(2+) influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd(3+)-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca(2+) influx by TNF-α, whereas potentiated TNF-α induced PGE(2) production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca(2+) influx, and COX-2-dependent PGE(2) production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE(2) production without affecting TRPC1 expression and the Ca(2+) influx. Finally, the suppression of store-dependent Ca(2+) influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE(2) production, respectively, at the downstream of TRPC1-associated Ca(2+) influx.

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P2-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P2 regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.

Quantitative measurement of Ca 2+-dependent calmodulin-target binding by Fura-2 and CFP and YFP FRET imaging in living cells

Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.

Regulation of cardiovascular TRP channel functions along the NO-cGMP-PKG axis

There is growing body of evidence that nitric oxide (NO)–cGMP–PKG signaling plays a central role in negative regulation of cardiovascular (CV) responses and its disorders through suppressed Ca2+ dynamics. Other lines of evidence also reveal the stimulatory effects of this signaling on some CV functions. Recently, transient receptor potential (TRP) channels have received much attention as non-voltage-gated Ca2+ channels involved in CV physiology and pathophysiology. Available information suggests that these channels undergo both inhibition and activation by NO via PKG-mediated phosphorylation and S-nitrosylation, respectively, and also act as upstream regulators to promote endothelial NO production. This review summarizes the roles of NO–cGMP–PKG signaling pathway, particularly in regulating TRP channel functions with their associated physiology and pathophysiology.

Tooth extraction in patients taking nonvitamin K antagonist oral anticoagulants

Background/purpose The nonvitamin K antagonist oral anticoagulants direct-thrombin inhibitor dabigatran and the Xa inhibitors rivaroxaban and apixaban are now being used clinically. The course of the patients on these anticoagulants who underwent tooth extraction was assessed. Materials and methods The medical charts of these patients were investigated. Tooth extraction was performed while maintaining conventional anticoagulant therapy. Results Twenty-three teeth were extracted in 19 patients, including two surgical extractions. Among the 19 patients, nine patients ingested rivaroxaban, six apixaban, and four dabigatran. One patient on rivaroxaban showed persistent postoperative bleeding following two surgical extractions. Mild oozing was observed in five patients (two on rivaroxaban and three on apixaban). There was no bleeding episode in the patients on dabigatran. Conclusion The patients on rivaroxaban with a prolonged prothrombin time value have a higher risk of bleeding, especially undergoing surgical extraction. Apixaban correlates to neither activated partial thromboplastin time nor prothrombin time values and the countermeasures should be employed based on the clinical findings.

Applying orthodontic tooth extrusion in a patient treated with bisphosphonate and irradiation: a case report

Bisphosphonates and irradiation are useful medical treatments, but can often cause oral complications such as medication-related oral necrosis of the jaw (MRONJ) and osteoradionecrosis (ORN) during oral surgery, including tooth extraction. Therefore, we should take all risks into consideration carefully before choosing dental treatment for patients with a medical history of such therapies. A 55-year-old woman who underwent cord blood transplantation to treat extranodal natural killer T (NK/T) cell lymphoma (nasal type IVB) had a medical history of bisphosphonate and irradiation treatments. We treated her residual tooth root by applying orthodontic extrusion to avoid extraction and successfully restored the tooth. Application of an orthodontic tooth extrusion technique for conservative treatment of a residual tooth is a useful means of avoiding MRONJ or ORN in patients who have a medical history of bisphosphonate and irradiation treatments.

Surface vacuolar ATPase in ameloblastoma contributes to tumor invasion of the jaw bone

Ameloblastoma is the most common benign odontogenic tumor in Japan. It is believed that it expands in the jaw bone through peritumoral activation of osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) released from the ameloblastoma, as in bone metastases of cancer cells. However, the clinical features of ameloblastoma, including its growth rate and patterns of invasion, are quite different from those of bone metastasis of cancer cells, suggesting that different underlying mechanisms are involved. Therefore, in the present study, we examined the possible mechanisms underlying the invasive expansion of ameloblastoma in the jaw bone. Expression levels of RANKL assessed by western blotting were markedly lower in ameloblastoma (AM-1) cells than in highly metastatic oral squamous cell carcinoma (HSC-3) cells. Experiments coculturing mouse macrophages (RAW264.7) with AM-1 demonstrated low osteoclastogenic activity, as assessed by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell formation, probably because of low release of RANKL, whereas cocultures of RAW264.7 with HSC-3 cells exhibited very high osteoclastogenic activity. Thus, RANKL release from AM-1 appeared to be too low to generate osteoclasts. However, AM-1 cultured directly on calcium phosphate-coated plates formed resorption pits, and this was inhibited by application of bafilomycin A1. Furthermore, vacuolar-type H+-ATPase (V-ATPase) and H+/Cl- exchange transporter 7 (CLC-7) were detected on the surface of AM-1 cells by plasma membrane biotinylation and immunofluorescence analysis. Immunohistochemical analysis of clinical samples of ameloblastoma also showed plasma membrane-localized V-ATPase and CLC-7 in the epithelium of plexiform, follicular and basal cell types. The demineralization activity of AM-1 was only 1.7% of osteoclasts demineralization activity, and the growth rate was 20% of human normal skin keratinocytes and HSC-3 cells. These results suggest that the slow expansion of several typical types of ameloblastomas in jaw bone is attributable to its slow growth and low demineralization ability.