We investigated the possible involvement of the melastatin family protein TRPM7 in Ca2+-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca2+ concentration with a parallel increase in the magnitude of spontaneous Ca2+ influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (Ispont) having Ca2+/Mg2+-inhibited but -permeating properties. Various cation channel blockers inhibiting Ispont (Gd3+, La3+, LOE908, 2-APB) suppressed the spontaneous Ca2+ influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg2+ or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of Ispont. In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced Ispont density and the magnitude of spontaneous Ca2+ influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G1/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca2+ influx pathway.

Stretch-activated cation channels (SACs) have been observed in many types of smooth muscle cells. However, the molecular identity and activation mechanisms of SACs remain poorly understood. We report that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Negative pressure (≥20 mmHg, cell-attached mode) activated single channels (approximately 20 pS) in isolated CAMs. These channels were permeable to Na+ and Cs+ and inhibited by Gd3+ (30 μM) and DIDS (100 μM). The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca2+ (>100 nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Caffeine (5 mM), which depletes SR Ca2+-stores, first activated and then abolished SACs. Tetracaine (100 μM), a ryanodine receptor antagonist, inhibited SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels that were activated by both negative pressure and Ca2+ and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These studies indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, possibly through ryanodine receptor activation, and this may contribute to the depolarization and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation.

Mechanisms linked to actin filaments have long been thought to cooperate in smooth muscle contraction, although key molecules were unclear. We show evidence that cardiac troponin T (cTnT) substantially contributes to Ca²⁺-mediated contraction in a physiological range of cytosolic Ca²⁺ concentration ([Ca²⁺]_i). cTnT was detected in various smooth muscles of the aorta, trachea, gut and urinary bladder, including in humans. Also, cTnT was distributed along with tropomyosin in smooth muscle cells, suggesting that these proteins are ready to cause smooth muscle contraction. In chemically permeabilised smooth muscle of cTnT^+/− mice in which cTnT reduced to ~50%, the Ca²⁺-force relationship was shifted toward greater [Ca²⁺]_i, indicating a sizeable contribution of cTnT to smooth muscle contraction at [Ca²⁺]_i < 1 μM. Furthermore, addition of supplemental TnI and TnC reconstructed a troponin system to enhance contraction. The results indicated that a Tn/Tn-like system on actin-filaments cooperates together with the thick-filament pathway.

Epidermal growth factor receptor (EGFR) is highly expressed in several types of cancer cells including oral squamous cell carcinoma (OSCC). EGF/EGFR signaling is recognized as an important molecular target in cancer therapy. However, cancer cells often become tolerant to EGF/EGFR signaling-targeted therapies. In the tumor microenvironment, the tumor incites inflammation and the inflammation-derived cytokines make a considerable impact on cancer development. In addition, hyperosmolarity is also induced, but the role of osmotic stress in cancer development has not been fully understood. This study demonstrates molecular insights into hyperosmolarity effect on OSCC development and shows that NFAT5 transcription factor plays an important functional role in enhancing the oral cancer cell proliferation by inducing the EGFR translocation from the endoplasmic reticulum to the plasma membrane through increase the expression of DPAGT1, an essential enzyme for catalyzing the first committed step of N-linked protein glycosylation. These results suggest that hyperosmolarity-induced intra-nuclear translocation of NFAT5 essential for DPAGT1 activation and EGFR subcellular translocation responsible for OSCC tumor progression.

The effects of NO donors on Ca²⁺‐dependent Cl⁻ currents (I_Cl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole‐cell patch clamp technique. With nystatin‐perforated whole‐cell recording, carbachol (CCh, ≥ 1 μm) induced a transient inward current (I_CCh) with a reversal potential of about ‐20 mV. Activation of I_CCh probably occurred through the M₃ muscarinic receptor, since nanomolar concentrations of 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) greatly inhibited this current, while 11‐(2‐(diethylamino)methyl)‐1‐piperidinylacetyl)‐5,11‐dihydro‐6H‐pyrido (2,3β) (1,4)benzodiazepine‐6‐one (AF‐DX 116) or pirenzepine at concentrations of up to 1 μm were almost ineffective. Chloride channel/transporter blockers such as DIDS (100 μm), anthracene‐9‐carboxylic acid (9‐AC, 100 μm) and niflumic acid (100 μm) greatly inhibited I_CCh, but cation channel blockers, such as nifedipine (10 μm), Zn²⁺ (500 μm) or Gd³⁺ (500 μm), were without effect. Activation of I_CCh was strongly attenuated by pretreatment with ryanodine (4 μm) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml⁻¹) in the pipette abolished a substantial part of I_CCh. These results suggest that I_CCh is I_Cl(Ca), which is activated by inositol 1,4,5‐trisphosphate (IP₃)‐mediated Ca²⁺ release. The nitric oxide donor S‐nitroso‐N‐acetyl penicillamine (SNAP) reduced the amplitude of I_CCh dose dependently (IC₅₀, ≈10 μm). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (±)‐E‐methyl‐2‐(E‐hydroxyimitol)‐5‐nitro‐6‐methoxy‐3‐hexeneamide (NO‐R). SNAP‐induced I_CCh inhibition was effectively antagonized by Methylene Blue (1‐100 nM), and mimicked by dibutyryl cGMP (db‐cGMP) (0.5‐1 mM), whereas two structurally distinct types of cGMP‐dependent (G)‐kinase inhibitor, N‐(2‐aminoethyl)‐5‐isoquinilinesulphonamide (H‐8, 2.5 μm) and KT5823 (1 μm), failed to counteract the inhibitory effects of SNAP or db‐cGMP. Another G‐kinase‐specific inhibitor R_p‐8‐(para‐chlorophenylthio)guanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cGMPS; 1 μm) itself caused a marked reduction in I_CCh. SNAP (100 μm) or db‐cGMP (100 μm) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP₃ were used instead of CCh to activate the inward current. These results suggest that inhibition of I_CCh by NO donors involves a cGMP‐dependent but G‐kinase‐independent mechanism, which may operate at a site(s) between the muscarinic (M₃) and IP₃ receptors.

Ameloblastoma (AB) is the most common benign epithelial odontogenic tumor occurring in the jawbone. AB is a slowly growing tumor but sometimes shows a locally invasive and an aggressive growth pattern with a marked bone resorption. In addition, the local recurrence and distant metastasis of AB also sometimes occurs, which resembles one of the typical malignant potentials. From these points of view, to understand better the mechanisms of AB cell migration or invasion is necessary for the better clinical therapy and improvements of the patients’ quality of life. Microtubules in eukaryotic cells reveal the shape of hollow cylinders made up of polymerized alpha (α)- and beta (β)-tubulin dimers and form the cytoskeleton together with microfilaments and intermediate filaments. Microtubules play important roles in cell migration by undergoing assembly and disassembly with post-translational modifications. Stability of microtubules caused by their acetylation is involved in cell migration. In this study, we investigated the expression and distribution of acetylated α-tubulin and alpha-tubulin N-acetyltransferase 1 (αTAT1), an enzyme which acetylates Lys-40 in α-tubulin, in AB specimens, and analyzed how tubulin was acetylated by αTAT1 activation in a human AB cell line, AM-1. Finally, we clarified that TGF-β-activated kinase1 (TAK1) was phosphorylated by TGF-β stimulation, then, induced tubulin acetylation via αTAT1 activation, which subsequently activated the migration and invasion of AB cells.