The Hippo pathway noncanonically drives autophagy and cell survival in response to energy stress.

The Hippo pathway is known for its crucial involvement in development, regeneration, organ size control, and cancer. While energy stress is known to activate the Hippo pathway and inhibit its effector YAP, the precise role of the Hippo pathway in energy stress response remains unclear. Here, we report a YAP-independent function of the Hippo pathway in facilitating autophagy and cell survival in response to energy stress, a process mediated by its upstream components MAP4K2 and STRIPAK. Mechanistically, energy stress disrupts the MAP4K2-STRIPAK association, leading to the activation of MAP4K2. Subsequently, MAP4K2 phosphorylates ATG8-family member LC3, thereby facilitating autophagic flux. MAP4K2 is highly expressed in head and neck cancer, and its mediated autophagy is required for head and neck tumor growth in mice. Altogether, our study unveils a noncanonical role of the Hippo pathway in energy stress response, shedding light on this key growth-related pathway in tissue homeostasis and cancer.

Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity.

OBJECTIVE: The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. METHODS: Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. RESULTS: Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dosedependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. CONCLUSION: Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

Effect of long-chain inorganic polyphosphate treated with wheat phytase on interleukin 8 signaling in HT-29 cells.

OBJECTIVE: This study was performed to investigate the potential effect of wheat phytase on long-chain inorganic polyphosphate (polyP)-mediated interleukin 8 (IL-8) signaling in an intestinal epithelial cell line, HT-29 cells. METHODS: Cell viability and the release of the pro-inflammatory cytokine IL-8 in HT-29 cells exposed to polyP1150 (average of 1,150 phosphate residues) treated with or without wheat phytase were measured by the EZ-CYTOX kit and the IL-8 ELISA kit, respectively. Also, the activation of cellular inflammatory factors NF-κB and MAPK (p38 and ERK 1/2) in HT-29 cells was investigated using ELISA kits. RESULTS: PolyP1150 negatively affected the viability of HT-29 cells in a dose-dependent manner. However, 100 mM polyP1150 dephosphorylated by wheat phytase increased cell viability by 1.4-fold over that of the intact substrate. Moreover, the 24 h exposure of cells to enzyme-treated 50 mM polyP1150 reduced the secretion of IL-8 and the activation of NF-κB by 9% and 19%, respectively, compared to the intact substrate. PolyP1150 (25 and 50 mM) dephosphorylated by the enzyme induced the activation of p38 MAPK via phosphorylation to 2.3 and 1.4-fold, respectively, compared to intact substrate, even though it had little effect on the expression of ERK 1/2 via phosphorylation. CONCLUSION: Wheat phytase could attenuate polyP1150-induced IL-8 release in HT-29 cells through NF-κB, independent of MAP kinases p38 and ERK. Thus, wheat phytase may alleviate inflammatory responses including hypercytokinemia caused by bacterial polyP infection in animals. Therefore, wheat phytase has the potential as an anti-inflammatory therapeutic supplement in animal husbandry.

Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide.

The objective of this study was to characterize the enzymatic hydrolysis of lipopolysaccharide (LPS) by wheat phytase and to investigate the effects of wheat phytase-treated LPS on in vitro toxicity, cell viability and release of a pro-inflammatory cytokine, interleukin (IL)-8 by target cells compared with the intact LPS. The phosphatase activity of wheat phytase towards LPS was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine. In vitro toxicity of LPS hydrolyzed with wheat phytase in comparison to intact LPS was assessed. Cell viability in human aortic endothelial (HAE) cells exposed to LPS treated with wheat phytase in comparison to intact LPS was measured. The release of IL-8 in human intestinal epithelial cell line, HT-29 cells applied to LPS treated with wheat phytase in comparison to intact LPS was assayed. Wheat phytase hydrolyzed LPS, resulting in a significant release of inorganic phosphate for 1 h (p < 0.05). Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase. Wheat phytase effectively reduced the in vitro toxicity of LPS, resulting in a retention of 63% and 54% of its initial toxicity after 1-3 h of the enzyme reaction, respectively (p < 0.05). Intact LPS decreased the cell viability of HAE cells. However, LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cell to 14% (p < 0.05) when compared with intact LPS. In conclusion, wheat phytase is a potential therapeutic candidate and prophylactic agent for control of infections induced by pathogenic Gram-negative bacteria and associated LPS-mediated inflammatory diseases in animal husbandry.

Potential immune-modulatory effects of wheat phytase on the performance of a mouse macrophage cell line, Raw 264.7, exposed to long-chain inorganic polyphosphate.

OBJECTIVE: This experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP. METHODS: Nitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit. RESULTS: P700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 µM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700. CONCLUSION: In animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

Catalytic properties of wheat phytase that favorably degrades long-chain inorganic polyphosphate.

OBJECTIVE: This study was conducted to determine catalytic properties of wheat phytase with exopolyphosphatase activity toward medium-chain and long-chain inorganic polyphosphate (polyP) substrates for comparative purpose. METHODS: Exopolyphosphatase assay of wheat phytase toward polyP75 (medium-chain polyP with average 75 phosphate residues) and polyP1150 (long-chain polyP with average 1150 phosphate residues) was performed at pH 5.2 and pH 7.5. Its activity toward these substrates was investigated in the presence of Mg2+, Ni+2, Co2+, Mn2+, or EDTA. Michaelis constant (Km) and maximum reaction velocity (Vmax) were determined from Lineweaver-Burk plot with polyP75 or polyP1150. Monophosphate esterase activity toward pNPP (p-nitrophenyl phosphate) was assayed in the presence of polyP75 or polyP1150. RESULTS: Wheat phytase dephosphorylated polyP75 and polyP1150 at pH 7.5 more effectively than that at pH 5.2. Its exopolyphosphatase activity toward polyP75 at pH 5.2 was 1.4-fold higher than that toward polyP1150 whereas its activity toward polyP75 at pH 7.5 was 1.4-fold lower than that toward polyP1150. Regarding enzyme kinetics, Km for polyP75 was 1.4-fold lower than that for polyP1150 while Vmax for polyP1150 was 2-fold higher than that for polyP75. The presence of Mg2+, Ni+2, Co2+, Mn2+, or EDTA (1 or 5 mM) exhibited no inhibitory effect on its activity toward polyP75. Its activity toward polyP1150 was inhibited by 1 mM of Ni+2 or Co2+ and 5 mM of Ni+2, Co2+, or Mg2+. Ni+2 inhibited its activity toward polyP1150 the most strongly among tested additives. Both polyP75 and polyP1150 inhibited the monophosphate esterase activity of wheat phytase toward pNPP in a dose-dependent manner. CONCLUSION: Wheat phytase with an unexpected exopolyphosphatase activity has potential as a therapeutic tool and a next-generational feed additive for controlling long-chain polyP-induced inappropriate inflammation from Campylobacter jejuni and Salmonella typhimurium infection in public health and animal husbandry.