Yuka Itoh

Hypoxia-inducible factor-1α (HIF1α) switches on transient receptor potential ankyrin repeat 1 (TRPA1) gene expression via a hypoxia response element-like motif to modulate cytokine release.

Background: TRPA1 forms Ca2+- and Zn2+-permeable ion channels that sense noxious substances. Results: TNF-α and IL1-α induce TRPA1 gene expression via nuclear factor-κB signaling and downstream activation of HIF1α. Conclusion: HIF1α links inflammatory mediators to ion channel expression. Significance: HIF1α acts by binding to a specific hypoxia response element-like motif and its flanking regions in the TRPA1 gene. Transient receptor potential ankyrin repeat 1 (TRPA1) forms calcium (Ca2+)- and zinc (Zn2+)-permeable ion channels that sense noxious substances. Despite the biological and clinical importance of TRPA1, there is little knowledge of the mechanisms that lead to transcriptional regulation of TRPA1 and of the functional role of transcriptionally induced TRPA1. Here we show induction of TRPA1 by inflammatory mediators and delineate the underlying molecular mechanisms and functional relevance. In human fibroblast-like synoviocytes, key inflammatory mediators (tumor necrosis factor-α and interleukin-1α) induced TRPA1 gene expression via nuclear factor-κB signaling and downstream activation of the transcription factor hypoxia-inducible factor-1α (HIF1α). HIF1α unexpectedly acted by binding to a specific hypoxia response element-like motif and its flanking regions in the TRPA1 gene. The induced TRPA1 channels, which were intrinsically activated by endogenous hydrogen peroxide and Zn2+, suppressed secretion of interleukin-6 and interleukin-8. The data suggest a previously unrecognized HIF1α mechanism that links inflammatory mediators to ion channel expression.

17β-Estradiol Induces IL-1α Gene Expression in Rheumatoid Fibroblast-Like Synovial Cells through Estrogen Receptor α (ERα) and Augmentation of Transcriptional Activity of Sp1 by Dissociating Histone Deacetylase 2 from ERα

Rheumatoid arthritis (RA) occurs four times more frequently in women than in men, although the mechanistic basis of the gender difference is unknown. RA is characterized by the overproliferation of synoviocytes producing proinflammatory cytokines such as IL-1, implicated in the pathogenesis of the disease. In this study we examined whether 17β-estradiol (E2) induced IL-1α mRNA expression in the rheumatoid fibroblast-like cell line MH7A, as well as in primary synovial cells from RA patients, and investigated the underlying molecular mechanisms. E2 induced IL-1α mRNA expression in both cell types in an estrogen receptor-dependent manner. In MH7A cells ERα but not ERβ mediated the effects of E2. Deletion and mutation analysis revealed that a GC-rich region within the IL-1α gene promoter was responsible for the response to E2. EMSAs showed that Sp1 and Sp3 bound to the GC-rich region and that the transcriptional activity of Sp1 was up-regulated by the treatment with E2. Sp1 and ERα interacted physically regardless of the presence of E2. Physical interaction was also observed between ERα and histone deacetylase 2 (HDAC2), and E2 induced the dissociation of HDAC2 from ERα. These results suggest that E2 induces the dissociation of corepressor HDAC2 from ERα, which leads to the augmentation of Sp1 transcriptional activity through the GC-rich region within the IL-1α gene promoter.

An environmental sensor, TRPV4 is a novel regulator of intracellular Ca2+ in human synoviocytes

The activation of a vanilloid type 4 transient receptor potential channel (TRPV4) has an obligatory role in regulation of intracellular Ca(2+) (Ca(2+)(i)) in several types of cells including vascular and sensory organs. In this study, we provide evidence that TRPV4 is a functional regulator of Ca(2+)(i) in human synoviocytes. Although significant expression of TRPV4 in synoviocytes from patients with (RA) and without (CTR) rheumatoid arthritis was detected at mRNA and protein level, those in the human fibroblast-like synoviocyte line MH7A were rather lower. Consistently, the selective TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alphaPDD) effectively elevated Ca(2+)(i) in the RA and CTR cells, which was abolished by the removal of external Ca(2+). Moreover, the elevation was inhibited by ruthenium red, a blocker of TRPVs. In MH7A cells transfected with human TRPV4 (MH7A-V4), 4alphaPDD elevated the Ca(2+)(i) in a similar manner to those in the RA and CTR cells. Electrophysiological analysis also revealed that 4alphaPDD activated nonselective cationic currents in RA cells. Application of 227 mosM solution to the RA and MH7A-V4 cells elevated their Ca(2+)(i), but this does not occur when it was applied to MH7A cells. Treatment of RA but not MH7A cells with 4alphaPDD for 24 h reduced their production of IL-8. These results suggest that an environmental sensor, TRPV4, is a novel regulator of intracellular Ca(2+) in human synoviocytes.

Cigarette Smoke Condensate Upregulates the Gene and Protein Expression of Proinflammatory Cytokines in Human Fibroblast-Like Synoviocyte Line

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce proinflammatory cytokines, which are implicated in the pathogenesis of RA. When human fibroblast-like synoviocytes line MH7A was treated with cigarette smoke condensate (CSC), either mainstream or sidestream, expression levels of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, and CYP1A1 mRNA were upregulated in both time- and dose-dependent manners. The upregulatory effects of CSC on these cytokines were not significantly inhibited by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, suggesting that the effects of CSC were independent of AhR. Cycloheximide treatment indicated that the augmenting effect of CSC on IL-1alpha, IL-1beta and IL-8, but not IL-6 and CYP1A1, mRNA expression requires de novo protein synthesis. CSC also induced cytokines at protein levels and further augmented the effects of tumor necrosis factor alpha on induction of these cytokines at both mRNA and protein levels. These results support the epidemiological studies indicating a strong association between heavy cigarette smoking and pathogenesis of RA.

Cardiac fibroblasts have functional TRPV4 activated by 4α-phorbol 12,13-didecanoate

AIMS Vanilloid type transient receptor potential channel (TRPV) could be a potential environmental sensor to multiple stimuli in many types of cells. In this study, we provide the first evidence of functional vanilloid type 4 transient receptor potential channel (TRPV4) in rat cardiac fibroblasts (CFs). MAIN METHODS Expression of TRPV4 in CFs was analyzed at mRNA and protein level. Function of TRPV4 in CFs was evaluated using a selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alphaPDD) while measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane currents. KEY FINDINGS Analysis of expression of mRNA transcripts of TRPV subfamily revealed that TRPV2 and TRPV4 were expressed in CFs. Significant immunoreactivity to TRPV4 protein was also detected in CFs. When 4alphaPDD was applied to CFs, [Ca(2+)](i) was elevated in a concentration-dependent manner. The elevation of [Ca(2+)](i) was abolished by the removal of external Ca(2+) and by ruthenium red (RuR). 4alphaPDD also activated non-selective cation currents (NSCCs), which were suppressed by RuR. Moreover, pretreatment of CFs with short interference RNA (siRNA) targeting TRPV4 significantly reduced both 4alphaPDD-induced elevation of [Ca(2+)](i) and NSCC. SIGNIFICANCE These results provide strong evidence that endogenous TRPV4 functions as an important regulator of [Ca(2+)](i) in CFs.

TRPV4 partially participates in proliferation of human brain capillary endothelial cells.

AIMS The vanilloid type 4 transient receptor potential channel (TRPV4) is a potential environmental sensor to multiple stimuli in many types of cells. In this study, we show that TRPV4 activated by 4α-phorbol 12,13-didecanoate (4αPDD) and hypo-osmotic stimulation (HOS) is a regulator of intracellular calcium ([Ca(2+)](i)) in human brain capillary endothelial cells (HBCEs), and its activation can partially regulate cell proliferation of HBCEs. MAIN METHODS The expression of TRPV4 in HBCEs was analyzed at the mRNA and protein levels. The function of TRPV4 in HBCEs was evaluated using a TRPV4 agonist, 4αPDD, and HOS while measuring [Ca(2+)](i) and membrane currents. KEY FINDINGS Analysis of the mRNA transcripts of the TRPV subfamily revealed that TRPV2 and TRPV4 were expressed in HBCEs. Immunoreactivity to the TRPV4 protein was also detected in HBCEs, which were positively stained by von Willebrand factor and CD31. When 4αPDD was applied, [Ca(2+)](i) in HBCEs was elevated in a concentration-dependent manner. In addition, exposure of HBCEs to HOS at 228mOsm induced an elevation of [Ca(2+)](i). Application of 4αPDD also activated non-selective cation currents (NSCCs). Pretreatment of HBCEs with short interference RNA targeting TRPV4 (siRNA) significantly reduced the 4αPDD-induced elevation of [Ca(2+)](i). When HBCEs were treated for 24h with concentrations of 4αPDD between 0.3 and 3 μM, the cell proliferation was potentiated in a concentration-dependent manner. The potentiation was partially inhibited in HBCEs treated with siRNA. SIGNIFICANCE These data suggest that endogenous TRPV4, which functions as a regulator of [Ca(2+)](i) in HBCEs, partially controls cell proliferation.

The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor

Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX.

TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription

Transforming growth factor β (TGF-β) signaling facilitates tumor development during the advanced stages of tumorigenesis, but induces cell-cycle arrest for tumor suppression during the early stages. However, the mechanism of functional switching of TGF-β is still unknown, and it is unclear whether inhibition of TGF-β signaling results amelioration or exacerbation of cancers. Here we show that the tumor suppressor p53 cooperates with Smad proteins, which are TGF-β signal transducers, to selectively activate plasminogen activator inhibitor type-1 (PAI-1) transcription. p53 forms a complex with Smad2/3 in the PAI-1 promoter to recruit histone acetyltransferase CREB-binding protein (CBP) and enhance histone H3 acetylation, resulting in transcriptional activation of the PAI-1 gene. Importantly, p53 is required for TGF-β-induced cytostasis and PAI-1 is involved in the cytostatic activity of TGF-β in several cell lines. Our results suggest that p53 enhances TGF-β-induced cytostatic effects by activating PAI-1 transcription, and the functional switching of TGF-β is partially caused by p53 mutation or p53 inactivation during cancer progression. It is expected that these findings will contribute to optimization of TGF-β-targeting therapies for cancer.

The CDK inhibitor p21 is a novel target gene of ATF4 and contributes to cell survival under ER stress

Activating transcription factor 4 (ATF4) is well known for its role in the endoplasmic reticulum (ER) stress response. ATF4 also transcriptionally induces multiple effectors that determine cell fate depending on cellular context. In addition, ATF4 can communicate both pro‐apoptotic and pro‐survival signals. How ATF4 mediates its prosurvival roles, however, requires further investigation. Here, we report that the CDK inhibitor p21 is a novel target gene of ATF4. We identified two ATF4‐responsive elements, one of which directly binds ATF4, within the first intron of the p21 gene. Importantly, overexpression of p21 enhances cell survival following ER stress induction, while p21 knockdown increases cell death. These results suggest that p21 induction plays a vital role in the cellular response to ER stress and indicate that p21 is a prosurvival effector of ATF4.

Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells.

Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.