Mitogen-activated protein kinase Hog1 is activated in response to curcumin exposure in the budding yeast Saccharomyces cerevisiae
© Azad et al.; licensee BioMed Central. 2014
Received: 29 July 2014
Accepted: 5 December 2014
Published: 19 December 2014
Curcumin (CUR), an active polyphenol derived from the spice turmeric, has been traditionally used for centuries in ancient Indian medicine to treat a number of diseases. The physiological effects of CUR have been shown to be diverse; however, the target molecules and pathways that CUR affects have yet to be fully described.
Here, we demonstrate for the first time that the budding yeast mitogen-activated protein kinase (MAPK) Hog1 is essential for the response to CUR. Moreover, CUR-induced Hog1 phosphorylation was rescued by supplementation of iron to the growth medium. Hog1 was rapidly phosphorylated upon CUR treatment, but unlike the response to hyperosmotic shock (0.8 M NaCl), it remains activated for an extended period of time. A detailed analysis of HOG pathway mutants revealed that Pbs2p, Ptc2p, and Ssk2p are required for optimal CUR-induced Hog1 phosphorylation. We also observed a Hog1 dependent transcriptional response to CUR treatment that involved the up-regulation of glycerol-3-phosphate dehydrogenase 1 (GPD1), a factor that is essential for the hyperosmotic stress response.
Our present finding revealed the role of Hog1 MAPK in regulation of CUR-induced transcriptional response. We anticipate that our finding will enhance the understanding on the molecular mode of action of CUR on S. cerevisiae.
Yeast cells have evolved sophisticated mechanisms to withstand a variety of stresses including limited availability of nutrients, fluctuations in temperature, changes in osmolarity, and the presence of harmful agents such as radiation or toxic chemicals. A myriad of strategies have evolved to maintain cellular homeostasis under stressful conditions including the activation of mitogen-activated protein kinase (MAPK) pathways. Thus far, 5 MAPK pathways have been characterized in S. cerevisiae. The fundamental function of these MAPK pathways is to regulate gene expression in response to various extracellular signals.
The high osmolarity glycerol (HOG) pathway is one of the most thoroughly studied MAPK pathways in yeast. The HOG pathway involves the MAPK Hog1 that specifically responds to increased extracellular osmolarity and is essential for cell survival under these conditions. Yeast cells respond to osmotic stress by activating Hog1 phosphorylation and, subsequently, translocating Hog1 to the nucleus where it directly interacts with several transcription factors to modulate gene expression. Recently, several studies have demonstrated additional functions of the HOG MAPK pathway. Evidence shows that the HOG pathway is essential for regulating the stress adaptation response induced by heat , citric acid , or low temperature . The HOG pathway is also involved in providing tolerance to methylglyoxal  and the bacterial endotoxin lipopolysaccharide (LPS) , and reportedly plays a role in cell wall maintenance  and the distribution of proteins within the Golgi .
Curcumin (diferuloylmethane) is the principal bioactive agent in the spice turmeric . Turmeric contains a class of compounds known as the curcuminoids, which includes curcumin, desmethoxycurcumin and bisdesmethoxycurcumin . CUR has been consumed as a dietary supplement for centuries and has been widely used in ayurvedic medicines . Because of the promising therapeutic potential of CUR, several clinical trials have been initiated or conducted to explore the effect of dietary CUR in the prevention of neurodegenerative diseases, several forms of cancer including colon and pancreatic cancer, bowel diseases, and other diseases -. Although extensive research has been performed on this drug, new biological targets of CUR are still being identified. In previous work, we demonstrated that the medicinal properties of CUR are largely the result of its cumulative effect on iron starvation and epigenetic modifications . Thus, the present study was designed to test whether the Hog1 MAPK is also activated in yeast cells exposed to the natural compound CUR or not.
We demonstrate that CUR exposure in yeast cells leads to phosphorylation of Hog1 and up-regulation of GPD1 mRNA levels. The findings presented here strongly indicate that the ability of CUR to induce the osmoresponse may underlie many of the therapeutic activities of CUR.
Reagents and yeast strains
List of yeast strains used in this study
MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1
Erin K O’Shea lab
EYO 690, Hog1-GFP(His) Nhp6a-RFP (KanMX6) MATa
Erin K O’Shea lab
MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0
Yeast deletion collection-Open Biosystems (YDC-OB)
Isogenic to BY4743 hog1Δ::KANMX4
Isogenic to BY4743 pbs2Δ::KANMX4
Isogenic to BY4743 ssk1Δ::KANMX4
Isogenic to BY4743 sho1Δ ::KANMX4
Isogenic to BY4743 msb2Δ::KANMX4
Isogenic to BY4743 ssk2Δ::KANMX4
Isogenic to BY4743 ssk22Δ ::KANMX4
Isogenic to BY4743 ste50Δ::KANMX4
Isogenic to BY4743 ptc1Δ::KANMX4
Isogenic to BY4743 ptc2Δ ::KANMX4
Isogenic to BY4743 ptc3Δ::KANMX4
FACS analysis of yeast cells
FACS analysis was performed as described earlier ,. Briefly, yeast cells in the exponential growth phase were treated with alpha-factor to synchronize them in the G1 phase. Cells were released into media containing DMSO (control) or CUR (50 or 100 μM) at regular intervals for 6 h. Samples were collected and harvested by centrifugation. Ethanol was added to the cell pellets and they were vigorously vortexed. Samples were then centrifuged and washed once with 50 mM sodium citrate buffer (pH 7.0). RNase A was added to the samples and they were incubated at 37°C for 1 h. RNase A-treated samples containing 20 mg/ml propidium iodide (Sigma) were transferred to a BD FACS flow cytometer. DNA was detected using a BD FACS Aria III and analyzed using BD FACS Diva software.
Nuclear-cytosolic extracts preparation
The spheroplasts were made from yeast cells as described earlier . spheroplasts were washed once with ice-chilled wash buffer (100 mM KCl, 50 mM HEPES-KOH pH 7.5, 2.5 mM MgCl2, and 0.4 M Sorbitol) and lysed in lysis buffer (20mMHEPES at pH7.5, 50mMNaCl, 1mMEDTA, 0.1%Tween 20, 1mM phenylmethylsulphonyl fluoride, protease inhibitor cocktail) with 8 strokes in a chilled tight-fitting pestle dounce homogenizer, after 15min incubation on ice. Unbroken cells and debris were removed by centrifugation for 5min at 300g at 4°C. The cytoplasmic fraction (supernatant) was collected after a spin at 13000g for 20 min at 4°C and the nuclear-enriched fraction (pellet) was washed once with lysis buffer before collection. Normalized volumes of cytoplasmic fraction and nuclei fraction were then subjected to western-blotting analysis.
Protein extraction and western blot analysis
Whole cell extracts from untreated and CUR-treated samples were prepared using the trichloroacetic acid (TCA) extraction method as described previously . Western blotting was conducted following protocol used previously ,. IRDye 700CW anti-rabbit IgG (diluted 1:15 000; LICOR Biosciences) was used as a secondary antibody. Blots were scanned using the Odyssey Infrared Imager (LI-COR Biosciences). The following primary antibodies were used: anti-GFP (Sigma, G1544), anti-p38/phospho-Hog1 (Cell Signaling, #92115), and polyclonal antibodies against recombinant yeast TATA binding protein (TBP) was raised in rabbit (Bhat Bio-tech India (P) Ltd.).
Isolation of total RNA and real-time PCR
List of primers used in the present study
Forward primer sequence (5′-3′)
Reverse primer sequence (5′-3′)
CUR induced growth arrest in yeast cells was rescued by iron supplementation
Hog1 is phosphorylated in response to curcumin treatment
Next, we were interested to analyze the time point kinetics of CUR-induced Hog1 phosphorylation. As a positive control, 0.8M NaCl treated samples were analyzed for Hog1 phosphorylation and as per reports - phosphorylation of Hog1 started very early and remained detectable for 30 min, after which it started to decrease (Figure 2C). While in case of CUR treatment, it was possible to detect phosphorylated Hog1 within 15 minutes of incubation. Interestingly, unlike NaCl, the CUR-induced phosphorylated Hog1 remained phosphorylated for an extended duration (at least 120 min) as revealed by western blotting (Figure 2C). Taken together, these data clearly suggest that CUR exposure induces Hog1 phosphorylation.
Iron supplementation rescues curcumin-induced Hog1 phosphorylation
Since, we have observed the phosphorylation of Hog1 in response to CUR exposure; next we were motivated to observe the effect of CUR on HOG1 deletion mutant. We arrested hog1 Δ cells with alpha-factor to arrest them in G1 phase as described in materials and methods. G1 arrested hog1 Δ cells were released in fresh media containing DMSO or media supplemented with 100 μM CUR. The results from the FACS analysis revealed that the DMSO-treated cells quickly moved to the G2 phase within 30 minutes of release from the alpha-factor arrest (Figure 3Ea), whereas 100 μM CUR treatment led to a delay in cell cycle progression (Figure 3Eb). While, the cell cycle progression was resumed in presence of curcumin after supplementation with iron (Figure 3Ec). The results revealed that hog1 Δ yeast mutant can also recover cell cycle arrest in presence of iron. The dose of iron used in this experiment (100μM) doesn’t affect cell cycle progression (Figure 3Ed). Altogether, these results indicate that CUR induced growth arrest in yeast cells can be rescued by iron supplementation.
SSK2, MSB2, and PTC2 deletions abrogate curcumin-induced Hog1 phosphorylation
CUR-induced phosphorylated Hog1 gets translocated to the nucleus leading to over-expression of GPD1 mRNA levels
The diverse pharmacological activities of CUR have been attributed to its actions on multiple cellular targets, either by interacting physically with the targets themselves or by modulating transcription factors, enzyme activity, or gene expression. In this study, we examined the requirement for a functional HOG pathway to cope with CUR-induced stress. If signaling through Hog1 is essential for its function under CUR-induced stress, Hog1 should be activated by phosphorylation of residues Thr174 and Tyr176, which is required for Hog1 activation. Using commercial antibodies raised against phosphorylated human p38, we were able to detect the phosphorylation of Hog1 protein after exposure of CUR (Figure 2A).
Yeast Hog1 is a stress-activated kinase that is thought to be activated exclusively by osmotic stress. However, recent studies have implicated the role of this MAPK in mediating tolerance to a variety of stress conditions including osmotic , oxidative , heat , arsenic , and citric acid stress . We have observed the phosphorylation of Hog1 in the presence of CUR (Figure 2A) and provided evidence for the activation of the HOG pathway in yeast. Hog1 activation has been most extensively characterized under conditions of hyperosmotic shock. The exposure of S. cerevisiae to high-osmolarity leads to a rapid (<30 min) and sustained (up to several hours depending on the severity of the conditions) phosphorylation of Hog1 protein -. To determine whether CUR induces a slow or a rapid response, phosphorylation of Hog1 was measured at intervals following the addition of 100 μM CUR. Addition of CUR induced a rapid increase in Hog1 phosphorylation, but unlike NaCl-induced Hog1 phosphorylation, it sustained for an extended period of time (Figure 2C). It has been well documented that curcumin antagonizes yeast growth by chelating iron . Previously, we have demonstrated that supplementation of iron rescued the cells from the growth arrest phenotype . Moreover, we also found a novel way to reset epigenetic marks back to their normal levels by iron supplementation in the presence of curcumin . Hence, we were curious to understand whether CUR-induced phosphorylation of Hog1 can be restored after iron supplementation. Interestingly, the level of phosphorylated Hog1 was reduced significantly in presence of iron (Figure 3D) suggesting that the activation of MAPK Hog1 by CUR is also dependent on its iron chelating property.
Hog1 is activated through a series of phosphorylation events involving the MAPK kinase Pbs2 and several other upstream factors ,. To test the role of upstream components of the HOG pathway in the cellular response to CUR, we monitored the phosphorylation of Hog1 in yeast strains carrying mutations in components of the HOG pathway. As shown in Figure 4A and B, the pbs2 Δ mutant was defective in Hog1 phosphorylation because it is the only immediate upstream kinase for Hog1. To test the role of upstream members of the HOG pathway in the cellular response to CUR, we monitored the phosphorylation of Hog1 in yeast strains carrying mutations in the components of the HOG pathway. Interestingly, we also observed Hog1 phosphorylation defects in SSK2, MSB2, and PTC2 deletion mutants, suggesting the requirement of these factors for optimal Hog1 phosphorylation in response to CUR treatment. The comprehensive CUR-induced growth inhibition analysis in the mutants incapable of phosphorylating Hog1 warrants future study, where methods outlined by the Clinical Laboratory Standards Institute (CLSI) M38-A  or the European Committee on Antimicrobial Susceptibility Testing (EUCAST)  can be used.
One of the main functions of the MAPK pathway is the regulation of transcriptional events in response to specific stimuli. Because of its phosphorylation, Hog1 gets translocated to the nucleus , where it targets various transcription factors, leading to upregulation of GPD1 mRNA levels . Our results also revealed that CUR-induced phosphorylated Hog1 migrates to nucleus (Figure 5A). We have also demonstrated that CUR exposure causes up-regulation of GPD1 (Figure 5B), which is required for the synthesis of a major osmolyte, glycerol. Elevated glycerol production is a prerequisite for the adaptation of S. cerevisiae to hyperosmotic stress . Interestingly, the expression of GPD1 in response to CUR treatment requires functional Hog1 protein (Figure 5B). Notably, to the best of our knowledge, for the first time, we report that the CUR exposure leads to Hog1 phosphorylation in S. cerevisiae and this is followed by the activation of GPD1 gene expression. The CUR has several molecular targets, other than Hog1. For example, CUR is known to target various cell signaling pathways such as JAK/STAT, Wnt/β-catanin, Notch, PI3K/PKB, AMPK, DNA damage checkpoint pathway ,- and many others. Here, we have identified Hog1 as an additional target of CUR. Considering Hog1 is central kinase in osmotic stress pathway, the activation of Hog1 upon CUR treatment indicates that CUR induces osmotic stress.
We thank Erin K. O’Shea for providing us hog1Δ and Hog1-GFP yeast strains. This work was financially supported by the Department of Biotechnology (DBT) and the Department of Science & Technology (DST), Govt. of India to RST. The Centre for Scientific and Industrial Research (CSIR), Govt. of India is acknowledged for providing fellowship support to GKA. Lab members are acknowledged for helpful discussions throughout the study.
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