MIF/SCL3A2 depletion inhibits the proliferation and metastasis of colorectal cancer cells via the AKT/GSK‐3β pathway and cell iron death

This study investigated the mechanisms of migration inhibitory factor (MIF) and solute carrier family 3 member 2 (SLC3A2) in colorectal cancer progression. The levels of MIF and SLC3A2 expression in cells were measured by RT‐qPCR. SW480 and SW620 cells were transfected with sh‐MIF and sh‐SLC3A2, respectively. MIF, SLC3A2, GPX4, E‐cadherin and N‐cadherin expression were detected by immunofluorescence (IF). CCK8 and Transwell assays were performed to detect cell proliferation and migration. Co‐immunoprecipitation (CoIP) was used to measure the binding activity of MIF and SLC3A2. Finally, a nude mouse tumorigenicity assay was used to confirm the functions of MIF and SLC3A2 in colorectal cancer. Results showed that the levels of MIF and SLC3A2 expression were up‐regulated in colorectal cancer cells. Inhibition of MIF or SLC3A2 expression prevented cell proliferation, migration, epithelial‐mesenchymal transition (EMT) and invasion. In addition, knockdown of MIF and SLC3A2 promoted iron death in SW480 and SW620 cells. CoIP results showed that MIF and SLC3A2 directly interact with each other. Knockdown of both MIF and SLC3A2 inhibited tumour growth and metastasis via the AKT/GSK‐3β pathway in vivo. The Akt/GSK‐3β pathway was found to participate in regulating MIF and SLC3A2 both in vivo and in vitro. MIF and SLC3A2 might be potential biomarkers for monitoring the treatment of colorectal cancer.     

The regulation of TNFα production after heat and endotoxin stimulation is dependent on Annexin-A1 and HSP70

Febrile temperatures can induce stress responses which protect cells from damage and can reduce inflammation during infections and sepsis. However, the mechanisms behind the protective functions of heat in response to the bacterial endotoxin LPS are unclear. We have recently shown that Annexin-1 (ANXA1)-deficient macrophages exhibited higher TNFα levels after LPS stimulation. Moreover, we have previously reported that ANXA1 can function as a stress protein. Therefore in this study, we determined if ANXA1 is involved in the protective effects of heat on cytokine levels in macrophages after heat and LPS. Exposure of macrophages to 42 °C for 1 h prior to LPS results in an inhibition of TNFα production, which was not evident in ANXA1^−/− macrophages. We show that this regulation involves primarily MYD88-independent pathways. ANXA1 regulates TNFα mRNA stability after heat and LPS, and this is dependent on endogenous ANXA1 expression and not exogenously secreted factors. Further mechanistic studies revealed the possible involvement of the heat shock protein HSP70 and JNK in the heat and inflammatory stress response regulated by ANXA1. This study shows that ANXA1, an immunomodulatory protein, is critical in the heat stress response induced after heat and endotoxin stimulation.     

β-cell Smad2 null mice have improved β-cell function and are protected from diet-induced hyperglycemia

Understanding signaling pathways that regulate pancreatic β-cell function to produce, store, and release insulin, as well as pathways that control β-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-β) signaling is involved in a broad range of β-cell functions. The canonical TGF-β signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-β/smad2 signaling in regulating mature β-cell proliferation and function using β-cell-specific smad2 null mutant mice. β-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased β-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.     

Influence of Temperature Stress on in Vitro Fertilization and Heat Shock Protein Synthesis in Maize (Zea mays L.) Reproductive Tissues

This study was conducted to investigate the response of maize (Zea mays) male and female mature reproductive tissues to temperature stress. We have tested the fertilization abilities of the stressed spikelets and pollen using in vitro pollination-fertilization to determine their respective tolerance to stress. The synthesis of heat shock proteins (HSPs) was also analyzed in male and female tissues using electrophoresis of ³⁵S-labeled proteins and fluorography, to establish a relationship between the physiological and molecular responses. Pollen, spikelets, and pollinated spikelets were exposed to selected temperatures (4, 28, 32, 36, or 40°C) and tested using an in vitro fertilization system. The fertilization rate is highly reduced when pollinated spikelets are exposed to temperatures over 36°C. When pollen and spikelets are exposed separately to temperature stress, the female tissues appear resistant to 4 hours of cold stress (4°C) or heat stress (40°C). Under heat shock conditions, the synthesis of a typical set of HSPs is induced in the female tissues. In contrast, the mature pollen is sensitive to heat stress and is responsible for the failure of fertilization at high temperatures. At the molecular level, no heat shock response is detected in the mature pollen.     

Interaction of heat and salt shock in cultured tobacco cells

Cultured tobacco cells (Nicotiana tabacum L. var Wisconsin-38) developed tolerance to otherwise nonpermissive 54°C treatment when heat-shocked at 38°C (2 h) but not at 42°C. Heat-shocked cells (38°C) exhibited little normal growth when the 54°C stress came immediately after heat shock and normal growth when 54°C stress was administered 8 hours after heat shock. Heat shock extended the length of time that the cells tolerated 54°C. Tobacco cells developed tolerance to otherwise lethal 2% NaCl treatment when salt-shocked (1.2% NaCl for 3 hours). The time course for salt tolerance development was similar to that of thermotolerance. Heat-shocked cells (38°C) developed tolerance of nonpermissive salt stress 8 hours after heat shock. Alternatively, cells heat-shocked at 42°C exhibited immediate tolerance to lethal salt stress followed by a decline over 8 hours. Radioactive methionine incorporation studies demonstrated synthesis of heat shock proteins at 38°C. The apparent molecular weights range from 15 to 115 kilodaltons with a protein complex in the 15 to 20 kilodalton range. Synthesis of heat shock proteins appeared to persist at 42°C but with large decreases in incorporation into selected heat shock protein. During salt shock, the synthesis of normal control proteins was reduced and a group of salt shock proteins appeared 3 to 6 h after shock. Similarities between the physiology and salt shock proteins/heat shock proteins suggest that both forms of stress may share common elements.     

Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts

Joint capsule fibrosis caused by excessive inflammation results in post-traumatic joint contracture (PTJC). Transforming growth factor (TGF)-β1 plays a key role in PTJC by regulating fibroblast functions, however, cytokine-induced TGF-β1 expression in specific cell types remains poorly characterized. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in inflammation- and fibrosis-associated pathophysiology. In this study, we investigated whether MIF can facilitate TGF-β1 production from fibroblasts and regulate joint capsule fibrosis following PTJC. Our data demonstrated that MIF and TGF-β1 significantly increased in fibroblasts of injured rat posterior joint capsules. Treatment the lesion sites with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP) reduced TGF-β1 production and relieved joint capsule inflammation and fibrosis. In vitro, MIF facilitated TGF-β1 expression in primary joint capsule fibroblasts by activating mitogen-activated protein kinase (MAPK) (P38, ERK) signaling through coupling with membrane surface receptor CD74, which in turn affected fibroblast functions and promoted MIF production. Our results reveal a novel function of trauma-induced MIF in the occurrence and development of joint capsule fibrosis. Further investigation of the underlying mechanism may provide potential therapeutic targets for PTJC.     

Mmi1, the Yeast Homologue of Mammalian TCTP,Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.     

Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n = 6 per group) were subjected to 90 min warm ischemia followed by 0.5 h, 6 h and 24 h reperfusion, respectively to liver transplantation (LTx) after 6 h of cold ischemia followed by 24 h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6 h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5 h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.     

Selective stimulation of Hsp27 and αB-crystallin but not Hsp70 expression by p38 MAP kinase activation

The levels of Hsp27 and αB-crystallin in C6 rat glioma cells, that had been heated at 43°C for 30 min with a subsequent culture for 16 h at 37°C, were markedly increased. The exposure of the cells to a low concentration (0.1–3 µg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and αB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and αB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1–3 µg/ml anisomycin. The results of a reporter assay, using an αB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of αB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 µg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO₂, or CdCl₂. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 µg/ml of anisomycin. The heat-induced accumulations of Hsp27 and αB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and αB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and αB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.     

Ocular blood flow decreases during passive heat stress in resting humans

Background

Heat stress induces various physiological changes and so could influence ocular circulation. This study examined the effect of heat stress on ocular blood flow.

Findings

Ocular blood flow, end-tidal carbon dioxide (P_ETCO₂) and blood pressure were measured for 12 healthy subjects wearing water-perfused tube-lined suits under two conditions of water circulation: (1) at 35°C (normothermia) for 30 min and (2) at 50°C for 90 min (passive heat stress). The blood-flow velocities in the superior temporal retinal arteriole (STRA), superior nasal retinal arteriole (SNRA), and the retinal and choroidal vessels (RCV) were measured using laser-speckle flowgraphy. Blood flow in the STRA and SNRA was calculated from the integral of a cross-sectional map of blood velocity. P_ETCO₂ was clamped at the normothermia level by adding 5% CO₂ to the inspired gas. Passive heat stress had no effect on the subjects’ blood pressures. The blood-flow velocity in the RCV was significantly lower after 30, 60 and 90 min of passive heat stress than the normothermic level, with a peak decrease of 18 ± 3% (mean ± SE) at 90 min. Blood flow in the STRA and SNRA decreased significantly after 90 min of passive heat stress conditions, with peak decreases of 14 ± 3% and 14 ± 4%, respectively.

Conclusion

The findings of this study suggest that passive heat stress decreases ocular blood flow irrespective of the blood pressure or arterial partial pressure of CO₂.     

Mouse liver is more resistant than skeletal muscle to heat-induced apoptosis

During passive heat stress, shifting of blood flow from the hepato-splanchnic to peripheral regions produces less favorable physiological conditions in the liver than in the skeletal muscle. We were wondering if the two organs differ in susceptibility to heat injury and thus examined the effects of heat shock exposure on apoptotic and heat stress-related markers in the gastrocnemius muscle and liver of mice. During heat exposure, mice had a peak core body temperature of 41.1 ± 0.7 °C. Heat-exposed mice showed higher levels of reactive oxygen species (ROS), cleaved caspases, fragmented DNA, and Drp1 protein expression in the gastrocnemius muscles than control mice. These changes were not observed in the livers of heat-exposed mice. Furthermore, the levels of glucocorticoid receptor, HSP70, and HSF1 proteins were significantly elevated in the gastrocnemius muscles of heat-exposed mice compared with that of control mice. The livers of heat-exposed mice also revealed increased expression of HSP70 but no changes in the other proteins. These results demonstrate that heat exposure induces significantly lower levels of the stress response and apoptosis in the liver than in the skeletal muscle of mice. The liver tissue resistance against heat stress is associated with low levels of heat-induced ROS production and mitochondrial fission protein expression.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum

In this study we examined the effect of heat stress (up to 6 hours at 43°C) on the biosynthesis and transport of phytohemagglutinin (PHA) in cotyledons of developing seeds of the common bean, Phaseolus vulgaris. Heat stress resulted in a decrease of total protein synthesis and an enhancement of the synthesis of heat shock proteins and PHA. Pulse chase experiments showed that a considerable proportion of the newly synthesized PHA was present in the endoplasmic reticulum (ER)/Golgi fraction and did not readily chase-out. Analysis with endoglycosidase H showed that the oligosaccharide sidechains of PHA were almost entirely in the high mannose configuration, indicating that most of the newly synthesized PHA was in the ER. However, some of the PHA became fucosylated at 43°C, indicating fucosyltransferase activity. That the biosynthesis and secretion of fucosyl-containing cell wall polymers proceeded normally at 43°C provided evidence that certain Golgi functions (i.e. transport to the cell wall) remained unaffected by heat stress. The ER obtained from these heat stress cotyledons had a greater density (1.16 g· cm⁻³ at 43°C instead of 1.14 g·cm⁻³ at 22°C) in sucrose gradients. Ultrastructural observations showed that the width of the lumen of the ER cisternae had increased from 20 nanometers at 22°C to 60 to 80 nanometers at 43°C; the lumen was filled with electrondense material presumed to be protein. The experiments are interpreted as evidence that heat stress imposes a block in the transport of PHA out of the ER. Whether heat stress affects the ER itself or alters the conformation of PHA, thereby preventing its transport, is not clear.     

The ω Subunit of RNA Polymerase Is Essential for Thermal Acclimation of the Cyanobacterium Synechocystis Sp. PCC 6803

The rpoZ gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions (constant illumination at 40 µmol photons m⁻² s⁻¹; 32°C; ambient CO₂) but was heat sensitive and died at 40°C. In the control strain, 71 genes were at least two-fold up-regulated and 91 genes down-regulated after a 24-h treatment at 40°C, while in ΔrpoZ 394 genes responded to heat. Only 62 of these heat-responsive genes were similarly regulated in both strains, and 80% of heat-responsive genes were unique for ΔrpoZ. The RNA polymerase core and the primary σ factor SigA were down-regulated in the control strain at 40°C but not in ΔrpoZ. In accordance with reduced RNA polymerase content, the total RNA content of mild-heat-stress-treated cells was lower in the control strain than in ΔrpoZ. Light-saturated photosynthetic activity decreased more in ΔrpoZ than in the control strain upon mild heat stress. The amounts of photosystem II and rubisco decreased at 40°C in both strains while PSI and the phycobilisome antenna protein allophycocyanin remained at the same level as in standard conditions. The phycobilisome rod proteins, phycocyanins, diminished during the heat treatment in ΔrpoZ but not in the control strain, and the nblA1 and nblA2 genes (encode NblA proteins required for phycobilisome degradation) were up-regulated only in ΔrpoZ. Our results show that the ω subunit of RNAP is essential in heat stress because it is required for heat acclimation of diverse cellular processes.     

Limited heat tolerance in an Arctic passerine: Thermoregulatory implications for cold‐specialized birds in a rapidly warming world

1. Arctic animals inhabit some of the coldest environments on the planet and have evolved physiological mechanisms for minimizing heat loss under extreme cold. However, the Arctic is warming faster than the global average and how well Arctic animals tolerate even moderately high air temperatures (T _a) is unknown.
2. Using flow‐through respirometry, we investigated the heat tolerance and evaporative cooling capacity of snow buntings (Plectrophenax nivalis; ≈31 g, N = 42), a cold specialist, Arctic songbird. We exposed buntings to increasing T _a and measured body temperature (T _b), resting metabolic rate (RMR), rates of evaporative water loss (EWL), and evaporative cooling efficiency (the ratio of evaporative heat loss to metabolic heat production).
3. Buntings had an average (±SD) T _b of 41.3 ± 0.2°C at thermoneutral T _a and increased T _b to a maximum of 43.5 ± 0.3°C. Buntings started panting at T _a of 33.2 ± 1.7°C, with rapid increases in EWL starting at T _a = 34.6°C, meaning they experienced heat stress when air temperatures were well below their body temperature. Maximum rates of EWL were only 2.9× baseline rates at thermoneutral T _a, a markedly lower increase than seen in more heat‐tolerant arid‐zone species (e.g., ≥4.7× baseline rates). Heat‐stressed buntings also had low evaporative cooling efficiencies, with 95% of individuals unable to evaporatively dissipate an amount of heat equivalent to their own metabolic heat production.
4. Our results suggest that buntings’ well‐developed cold tolerance may come at the cost of reduced heat tolerance. As the Arctic warms, and this and other species experience increased periods of heat stress, a limited capacity for evaporative cooling may force birds to increasingly rely on behavioral thermoregulation, such as minimizing activity, at the expense of diminished performance or reproductive investment.

     

Changes in RACK1 expression induce defects in nodulation and development in Phaseolus vulgaris

RACK1 is a scaffold protein with the ability to interact in a regulated manner with a diverse number of ligands from distinct signal-transduction pathways. This assessment allowed us to infer that it may be involved in different processes such as nodulation. In a recent study we showed by silencing, that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development in Phaseolus vulgaris. On the other hand, we have also observed that its overexpression provokes a dramatic phenotype in: (a) seedlings that have been exposed to heat, in which systemic necrosis is induced; and (b) in Agrobacterium rhizogenes-transformed roots, where nodulation is strongly inhibited and nodules show early senescent symptoms. These findings indicate that PvRACK1 may be an integrator of diverse signal-transduction pathways in processes as varied as nodulation, cell expansion, heat stress responses, and systemic activation of necrosis.