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Mechanical-loading and unloading can modify osteoblast functioning. Ca2+ signaling is one of the earliest events in osteoblasts to induce a mechanical stimulus, thereby demonstrating the importance of the underlying mechanical sensors for the sensation. Here, we examined the mechano-sensitive channels PIEZO1 and TRPV4 were involved in the process of mechano-sensation in the osteoblastic MC3T3-E1 cells. The analysis of mRNA expression revealed a high expression of Piezo1 and Trpv4 in these cells. We also found that a PIEZO1 agonist, Yoda1, induced Ca2+ response and activated cationic currents in these cells. Ca2+ response was elicited when mechanical stimulation (MS), with shear stress, was induced by fluid flow in the MC3T3-E1 cells. Gene knockdown of Piezo1 in the MC3T3-E1 cells, by transfection with siPiezo1, inhibited the Yoda1-induced response, but failed to inhibit the MS-induced response. When MC3T3-E1 cells were transfected with siTrpv4, the MS-induced response was abolished and Yoda1 response was attenuated. Moreover, the MS-induced response was inhibited by a TRPV4 antagonist HC-067047 (HC). Yoda1 response was also inhibited by HC in MC3T3-E1 cells and HEK cells, expressing both PIEZO1 and TRPV4. Meanwhile, the activation of PIEZO1 and TRPV4 reduced the proliferation of MC3T3-E1, which was reversed by knockdown of PIEZO1, and TRPV4, respectively. In conclusion, TRPV4 and PIEZO1 are distinct mechano-sensors in the MC3T3-E1 cells. However, PIEZO1 and TRPV4 modify the proliferation of these cells, implying that PIEZO1 and TRPV4 may be functional in the osteoblastic mechano-transduction. Notably, it is also found that Yoda1 can induce TRPV4-dependent Ca2+ response, when both PIEZO1 and TRPV4 are highly expressed.

Detection of mechanical stress is essential for diverse biological functions including touch, audition, and maintenance of vascular myogenic tone. PIEZO1, a mechano-sensing cation channel, is widely expressed in neuronal and non-neuronal cells and is expected to be involved in important biological functions. Here, we examined the possibility that PIEZO1 is involved in the regulation of synovial sarcoma cell-viability. Application of a PIEZO1 agonist Yoda1 effectively induced Ca2+ response and cation channel currents in PIEZO1-expressing HEK (HEK-Piezo1) cells and synovial sarcoma SW982 (SW982) cells. Mechanical stress, as well as Yoda1, induced the activity of an identical channel of conductance with 21.6 pS in HEK-Piezo1 cells. In contrast, Yoda1 up to 10 μM had no effects on membrane currents in HEK cells without transfecting PIEZO1. A knockdown of PIEZO1 with siRNA in SW982 cells abolished Yoda1-induced Ca2+ response and significantly reduced cell cell-viability. Because PIEZO1 is highly expressed in SW982 cells and its knockdown affects cell-viability, this gene is a potential target against synovial sarcoma.

The sesquiterpene (−)Englerin A (EA) is an organic compound from the plant Phyllanthus engleri which acts via heteromeric TRPC4/C1 channels to cause cytotoxicity in some types of cancer cell but not normal cells. Here we identified selective cytotoxicity of EA in human synovial sarcoma cells (SW982 cells) and investigated the mechanism. EA induced cation channel current (Icat) in SW982 cells with biophysical characteristics of heteromeric TRPC4/C1 channels. Inhibitors of homomeric TRPC4 channels were weak inhibitors of the Icat and EA-induced cytotoxicity whereas a potent inhibitor of TRPC4/C1 channels (Pico145) strongly inhibited Icat and cytotoxicity. Depletion of TRPC1 converted Icat into a current with biophysical and pharmacological properties of homomeric TRPC4 channels and depletion of TRPC1 or TRPC4 suppressed the cytotoxicity of EA. A Na + /K + -ATPase inhibitor (ouabain) potentiated EA-induced cytotoxicity and direct Na + loading by gramicidin-A caused Pico145-resistant cytotoxicity in the absence of EA. We conclude that EA has a potent cytotoxic effect on human synovial sarcoma cells which is mediated by heteromeric TRPC4/C1 channels and Na + loading.

Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold, mechanical stimulation, and irritant chemicals. In our recent study, 9, 10‐phenanthrenequinone (9,10‐PQ) is the most potent irritant for activation of NRF2 among 1395 cigarette smoke components and it may be, therefore, important to find its additional targets. Here, we show that 9,10‐PQ functions as an activator of TRPA1 in human embryonic kidney (HEK) cells expressing human wild‐type TRPA1 (HEK‐wTRPA1) and human alveolar A549 (A549) cells. Application of 9,10‐PQ at 0.1–10 μmol/L induced a concentration‐dependent Ca2+ response as well as inward currents at −50 mV in HEK‐wTRPA1 cells. The current response was blocked by TRPA1 antagonists, HC‐030031 (HC) and A‐967079. To test whether 9,10‐PQ affects the cysteine residues of TRPA1, we expressed mutant TRPA1 channels in HEK cells (HEK‐muTRPA1) in which six different cysteine residues were replaced with serine. Among them, a mutation of cysteine 621 (C621S) abolished the 9,10‐PQ‐induced Ca2+ and current responses. The channel activity induced by 9,10‐PQ was also abolished in excised inside‐out patches isolated from HEK‐muTRPA1 cells with the C621S substitution. Although a mutation of cysteine 665 (C665S) reduced the 9,10‐PQ‐induced response, channel sensitization by pretreatment with Cu2+ plus 1,10‐phenanthroline and by internal dialysis of 3 μmol/L Ca2+ restored the response. However, a double mutant with C621S and C665S substitutions had little response to 9,10‐PQ, even when sensitized by Ca2+ dialysis. In A549 cells, 9,10‐PQ induced an HC‐sensitive Ca2+ response. Our findings demonstrate that 9,10‐PQ activation of human TRA1 is dependent on cysteine residues 621 and 665.

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 μM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.

Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn2+ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3–100 nM of JT010 to HEK cells with hTRPA1 induced large Ca2+ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10–100 μM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1.

The effects of diclofenac (Dic), an acetic acid derivative‐type nonsteroidal anti‐inflammatory drug, were examined on the function of transient receptor potential (TRP) melastatin (TRPM) 3 (TRPM3) in human embryonic kidney 293 cell‐line (HEK293) cells with recombinant human TRPM3 isoforms (TRPM31325, TRPM3‐3, TRPM3‐9, and TRPM3‐S) and in a neuroblastoma cell line human neuroblastoma IMR‐32 cells (IMR‐32 cells) derived from human peripheral neurons. TRPM3 responses evoked by pregnenolone sulfate (PregS) were effectively inhibited by Dic in a concentration‐dependent manner in Ca2+ measurement and electrophysiological assays. The apparent IC50 for PregS‐induced Ca2+ response of TRPM31325, TRPM3‐3, and TRPM3‐9 was calculated to be 18.8, 42.5, and 7.1 μmol/L, respectively. The TRPM3‐dependent Ca2+ responses evoked by nifedipine, another TRPM3 agonist, were also significantly inhibited by Dic. In contrast, aceclofenac, an acetoxymethyl analog of Dic, had no effects on PregS‐induced TRPM3 responses. Constitutive channel activity of TRPM3‐S without TRPM3 agonists was substantially inhibited by Dic, ruling out the possibility of interaction of Dic against TRPM3 agonists to the channel binding sites. Moreover, Dic reversibly inhibited TRPM3 single‐channel activity recorded in excised outside‐out patches without affecting the channel conductance. In differentiated neuronal IMR‐32 cells with endogenous TRPM3, Dic inhibited PregS‐evoked Ca2+ responses with an apparent IC50 of 17.1 μmol/L. Taken together, our findings demonstrate that Dic inhibits human TRPM3 without interacting with the channel pore.

The effects of diclofenac (Dic), an acetic acid derivative‐type nonsteroidal anti‐inflammatory drug, were examined on the function of transient receptor potential ( TRP ) melastatin ( TRPM ) 3 ( TRPM 3) in human embryonic kidney 293 cell‐line ( HEK 293) cells with recombinant human TRPM 3 isoforms ( TRPM 3 1325 , TRPM 3‐3, TRPM 3‐9, and TRPM 3‐S) and in a neuroblastoma cell line human neuroblastoma IMR‐32 cells ( IMR ‐32 cells) derived from human peripheral neurons. TRPM 3 responses evoked by pregnenolone sulfate (PregS) were effectively inhibited by Dic in a concentration‐dependent manner in Ca 2+ measurement and electrophysiological assays. The apparent IC 50 for PregS‐induced Ca 2+ response of TRPM 3 1325 , TRPM 3‐3, and TRPM 3‐9 was calculated to be 18.8, 42.5, and 7.1  μ mol/L, respectively. The TRPM 3‐dependent Ca 2+ responses evoked by nifedipine, another TRPM 3 agonist, were also significantly inhibited by Dic. In contrast, aceclofenac, an acetoxymethyl analog of Dic, had no effects on PregS‐induced TRPM 3 responses. Constitutive channel activity of TRPM 3‐S without TRPM 3 agonists was substantially inhibited by Dic, ruling out the possibility of interaction of Dic against TRPM 3 agonists to the channel binding sites. Moreover, Dic reversibly inhibited TRPM 3 single‐channel activity recorded in excised outside‐out patches without affecting the channel conductance. In differentiated neuronal IMR ‐32 cells with endogenous TRPM 3, Dic inhibited PregS‐evoked Ca 2+ responses with an apparent IC 50 of 17.1  μ mol/L. Taken together, our findings demonstrate that Dic inhibits human TRPM 3 without interacting with the channel pore.

BackgroundAnastomotic leakage (AL) is one of the most serious complications after low anterior resection (LAR) for rectal cancer, and the significance of diverting stoma to prevent AL is still controversial. The aim of this study is to clarify the potential benefits and safety of diverting ileostomy (DI) following laparoscopic LAR in rectal cancer patients.MethodsThis was a retrospective cohort study of 417 rectal cancer patients who underwent laparoscopic LAR in a single institute. The risk factors for AL and the DI-related morbidity were assessed.ResultsDI was performed in 226 patients (54.2%). The incidence rates of symptomatic AL showed no significant difference between patients with and without DI (8.4% vs. 10.0%, p = 0.612). AL requiring a surgical intervention was relatively lower in patients with DI than in those without DI (1.8% vs. 4.7%, p = 0.097). DI construction was an independent risk factor for AL requiring a surgical intervention (OR 3.47, p = 0.041), as was the serum albumin level (p = 0.003), and being male was a relative risk factor (p = 0.058). Focusing on sex, the rate of AL requiring a surgical intervention was significantly different in male (1.7 and 7.9%, p = 0.021) but not in female patients (1.9 and 1.1%, p = 1.000) with and without DI. The DI construction-related morbidity was 9.7%, and no patient required a reoperation. Of 226 patients with DI, 209 (92.5%) underwent stoma closure 118 days (median 30–509 days) after LAR. The stoma closure-related morbidity was 9.1% and 1 patient (0.5%) required a reoperation due to anastomotic leakage.ConclusionsDI following laparoscopic LAR can decrease the risk of AL, requiring a surgical intervention, especially in male patients with malnutrition. However, due to DI-related morbidity, DI is not recommended in female patients.

In the Escherichia coli system catalysing oxidative protein folding, disulphide bonds are generated by the cooperation of DsbB and ubiquinone and transferred to substrate proteins through DsbA. The structures solved so far for different forms of DsbB lack the Cys104–Cys130 initial‐state disulphide that is directly donated to DsbA. Here, we report the 3.4 Å crystal structure of a DsbB–Fab complex, in which DsbB has this principal disulphide. Its comparison with the updated structure of the DsbB–DsbA complex as well as with the recently reported NMR structure of a DsbB variant having the rearranged Cys41–Cys130 disulphide illuminated conformational transitions of DsbB induced by the binding and release of DsbA. Mutational studies revealed that the membrane‐parallel short α‐helix of DsbB has a key function in physiological electron flow, presumably by controlling the positioning of the Cys130‐containing loop. These findings demonstrate that DsbB has developed the elaborate conformational dynamism to oxidize DsbA for continuous protein disulphide bond formation in the cell.