Expression of protein phosphatase 4 in different tissues under hypoxia

Yanyan Ma; Jing Hou; Dengliang Huang; Yaogang Zhang; Zhe Liu; Meiyuan Tian

doi:10.4103/ijpm.ijpm_1179_21

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Year : 2023 | Volume : 66 | Issue : 3 | Page : 577-583

Expression of protein phosphatase 4 in different tissues under hypoxia

Yanyan Ma¹, Jing Hou², Dengliang Huang², Yaogang Zhang², Zhe Liu², Meiyuan Tian¹
¹ Central Laboratory, Affiliated Hospital of Qinghai University, Tongren Road 29, Qinghai Province; Medical College of Qinghai University, Xining, Qinghai Province, China
² Central Laboratory, Affiliated Hospital of Qinghai University, Tongren Road 29, Qinghai Province, China

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Date of Submission	05-Dec-2021
Date of Decision	16-Jan-2022
Date of Acceptance	16-Feb-2022
Date of Web Publication	03-Feb-2023

Abstract

Relevant research data shows that there is a certain degree of energy metabolism imbalance in highland residents. Protein phosphatase 4 (PP4) has been found as a new factor in the regulation of sugar and lipid metabolism. Here, we investigate the differential expression of PP4 at a simulated altitude of 4,500 m in the heart, lung, and brain tissues of rats. A hypoxic plateau rat model was established using an animal decompression chamber. A blood routine test was performed by an animal blood cell analyzer on rats cultured for different hypoxia periods at 4,500 m above sea level. Quantitative polymerase chain reaction (qPCR) and western blot were used to detect the changes of protein phosphatase 4 catalytic subunit (PP4C) gene and protein in heart, lung, and brain tissues. The PP4C gene with the highest expression level found in rats slowly entering the high altitude area (20 m-2200 m-7 d-4500 m-3 d) was about twice as high as the low elevation group (20 m above sea level). The simulated high-altitude hypoxia induced an increase of PP4C expression level in all tissues, and the expression in the lung tissue was twice as expressed as heart and brain tissue at high altitude (P < 0.05). These results suggest that the PP4 phosphatase complex is ubiquitously expressed in rat tissues and likely involved in adaptation to or disease associated with high-altitude hypoxia.

Keywords: Brain tissue, heart tissue, hypoxia, lung tissue, protein phosphatase 4

How to cite this article:
Ma Y, Hou J, Huang D, Zhang Y, Liu Z, Tian M. Expression of protein phosphatase 4 in different tissues under hypoxia. Indian J Pathol Microbiol 2023;66:577-83

How to cite this URL:
Ma Y, Hou J, Huang D, Zhang Y, Liu Z, Tian M. Expression of protein phosphatase 4 in different tissues under hypoxia. Indian J Pathol Microbiol [serial online] 2023 [cited 2023 Sep 27];66:577-83. Available from: https://www.ijpmonline.org/text.asp?2023/66/3/577/369081

Introduction

Complex environments such as cold and hypoxia could induce severe health damage to people who are living in the high-altitude area in Qinghai-Tibetan Plateau for a long time period.^[1] When people from low altitude areas enter high altitude areas within a short period of time, a series of stress responses will take place in the body to adapt to the special environment. Physical changes are signs of adaptation to low-oxygen environments at high altitudes. Some people may be susceptible to low temperature, low pressure, and low oxygen environment, and different levels of altitude sickness may occur, which reduces people's tolerance and quality of life.^[2] The low-pressure and low-oxygen environment in high-altitude areas mainly affects the cardiopulmonary function of the human body, which is easy to cause acute altitude sickness in people entering high altitude regions.^[3] In severe cases, life-threatening diseases such as high-altitude pulmonary edema and high-altitude cerebral edema may even occur. A hypoxic environment can also cause hypoxia in brain tissue, leading to ischemic brain injury.^[4] Currently, it is the focus and frontier of high altitude medical research to actively search for the cause and reduce the reactions of altitude sickness. Relevant research data shows that there is a certain degree of imbalance in the energy metabolism of highland residents, which is one of the important causes of altitude sickness.^[5],[6]

Protein phosphatase 4 (PP4), an important member of the protein phosphatase PP2A family, has been discovered as a new factor in the regulation of sugar and lipid metabolism.^[7],[8],[9] Since the cloning of the PP4 gene in 1993, it has been shown to be involved in the regulation of many important cell processes.^[10] As a protein located in centrosomes, PP4 is involved in the maturation of centrosomes and growth of microtubules^[11]; PP4 also plays a role in DNA damage repair by dephosphorylation of Replication Protein A2 (RPA2) and H2AX Histone (H2AX).^[12] Furthermore, reconstitution of AMP-activated protein kinase (AMPK), or PP4 knockdown, enhances lipid clearance in mitochondrial calcium uniporter (MCU)Δ^hep hepatocytes. Recent studies have shown that an MCU/PP4/AMPK molecular cascade links Ca²⁺ dynamics to hepatic lipid metabolism.^[13]

However, although both high altitude diseases and PP4 are involved in cellular metabolism, few studies have investigated the role of PP4 in the heart, lung, and brain tissues that are most frequently affected under high-altitude conditions. In this study, changes in red blood cells and hemoglobin were systematically and dynamically observed in a rat model simulating a high altitude of 4,500 m, which to some extent indicated that the modeling was successful. The expression level of PP4 in the heart, lung, and brain tissues of the model rats was further explored, providing a clue of PP4 involvement in high-altitude diseases.

Materials and Methods

Main instruments and reagents

Animal decompression chamber (YanTai Bingchuan Hyperbaric Oxygen Chamber Co., LTD., DC0820), high-speed bench refrigerated centrifuge (Germany Sigma Company, 3-30k), fluorescence real-time quantitative polymerase chain reaction (qPCR) (Roche Light Cycler 480II), ultramicro nucleic acid protein tester (Nanodrop2000C), animal automatic blood cell analyzer(BC2800Vet), a chemiluminescent gel imaging system (AI600QC), PCR (LifeTechnologies Holding Pte Ltd, 9700), SYBR Green (Roche, 04913850001), Trizol (TaKaRa, 9109), proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) (BOSTER, AR1179), PP4 regulatory subunit 2 (PP4R2) antibody (ab70631) and protein phosphatase 4, catalytic subunit (PPX) antibody (ab16475) purchased from Abcam、Revertaid^TM First Strand cDNA Synthesis kit (One-Step gDNA Removal and cDNA Synthesis SuperMix TransScript)、PP4, and β-actin primer (synthesized by Shanghai Sangon Co., LTD.).

Animal model

The model used for this experiment was widely described and validated previously in several studies.^[14] The study was performed on 40 healthy male Wistar rats (8 weeks of age, weight, 250 ± 20 g) obtained from Qinglong Mountain animal breeding farm, Jiangning district, Nanjing city (license no.: 3201111979). Rats were placed into an animal decompression chamber (Yantai Ice Wheel High-Pressure Oxygen Chamber Co., Ltd.) to simulate low pressure and low oxygen conditions at high altitude areas. The animal decompression chamber parameter setting is as follows: simulated altitude of 4,500 m, lifting speed of 10 m/s, cabin pressure of 45 kPa, cabin oxygen pressure of 9.022 kpa, the operating time of the laboratory module is 24 h/d. Experiments were approved by the Ethics Committee of Affiliated Hospital of Qinghai University. Forty 8-week-old male Wistar rats were divided into the control group and three experimental groups. The control group was raised at an altitude of 20 m. The first experimental group was raised for 7 days in an animal decompression chamber at a simulated altitude of 4,500 m (20 m-4500 m-7 d). The second experimental group was first adapted to the altitude of 2200 m for 7 days and then placed in the animal decompression chamber at a simulated altitude of 4,500 m for 3 days (20 m-2200 m-7 d-4500 m-3 d). The third experimental group was first adapted to the altitude of 2200 m for 7 days and then placed in the animal decompression chamber at a simulated altitude of 4,500 m for 7 days (20 m-2200 m-7 d-4500 m-7 d). Animals were fed freely during animal decompression chamber feeding. The control group and the experimental groups were fed the same amount of food. At the end of the experiment, 3 ml of blood was taken from the heart of each rat immediately after anesthesia with 5% pentobarbital sodium. The heart, lung, and brain were then isolated and placed in a centrifuge tube sterilized by 0.1% diethylpyrocarbonate (DEPC) immersion, respectively, which was quickly transferred to an ultra-low temperature refrigerator at −80° for the extraction of total RNA and protein.

Detection of blood routine in rats

The blood sample was treated with ethylenediaminetetraacetic acid (EDTA) anticoagulant, and 20 ul of venous blood was mixed with the diluent and left at room temperature for 5 min. The red blood cell count (RBC), white blood cell count (WBC), lymphocyte count (Lymph), hemoglobin content (HGB), monocyte count (Mon), neutrophil count (Gran), platelet count (PLT), and mean corpuscular hemoglobin concentration (MCHC) were detected by animal blood cell analyzer. Each analysis was done at least in quintuplicate.

Rats, RNA extraction, and cDNA synthesis

All tissue samples were thoroughly rinsed by phosphate-buffered saline (PBS). Total RNA was extracted from the heart, lung, and brain tissue using the TRIzol reagent (TaKaRa) according to the manufacturer's instructions. Total RNA concentration and purity were measured by Nanodrop2000C (OD₂₆₀/OD₂₈₀ = 1.8 - 2.0). Total RNA mass was determined by 1.0% denatured agarose gel electrophoresis, and stored at −80°C until use. Single-strand cDNA was synthesized from about 3 ug of total RNA using the Revertaid^TM First Strand cDNA Synthesis kit (One-Step gDNA Removal and cDNA Synthesis SuperMix TransScript) with the random primer and was diluted 10-fold. The reverse transcriptional product is obtained and stored at −20°c.

Real-time qPCR analysis of PP4 gene expression in the heart, lung, and brain tissue

Filtered distilled water was used as the no-template control. Template without reverse transcription was used to check for contamination with genomic DNA. Protein phosphatase 4 catalytic subunit (PP4C) gene-specific primers, forward (5′- TGGCTGACTATTTCCTGTGAC-3′) and reverse (5′- CAACCTCTCCAGAGCAAGTC-3′), were designed to amplify a product of 146 bp, and the PCR product was sequenced to verify the specificity of the PCR primer. The rat β-actin primers (Forward: 5′-CCTCGTCTCATAGACAAGATGGT-3′; Reverse: 5′- GGGTAGAGTCATACTGGAACATG-3′) were used to amplify a 69 bp fragment of β-actin as a reference gene. The comparative cycle threshold (Ct) method was used to analyze the expression level of PP4. Each analysis was done at least in triplicate. The reaction system is 20 μl. The reaction conditions are as follows: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 60 s, annealing extension at 60°C for 34 s (fluorescence signal is collected in this step), 40 cycles (LightCycler 480II).

Western blot analysis

About each 100 mg heart, lung, and brain tissue preserved at −80°C was frozen with liquid nitrogen and then added with 0.5 ml radio Immunoprecipitation Assay Lysis buffer to extract protein. The same amount of total protein was taken from each sample, separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with PBS with a Tween-20 (sangon A100777) solution containing 5% skimmed milk. Goat anti-mouse PP4 antibody (1:200) and mouse anti- glyceraldehyde-3-phosphate dehydrogenase GAPDH (Proteintech, 60004-1-Ig) antibody (1:5000) were incubated with the PVDF membrane at room temperature for 2 h and the membrane was then washed. The membrane was then incubated for 2 h at room temperature with the corresponding second antibody (1:5000) and then washed again. The ECL Western Blotting KIT (sangon, C510043) was then used to stain the protein bands. Image J software was used to analyze the grayscale value of the target protein band and the internal reference within the protein electrophoresis image. The ratio of the two was used as the relative quantitative result for the target protein.

Data analysis

All data recorded were included in a database and analyzed using the Statistical Package for the Social Sciences (SPSS) program (IBM SPSS R V.21.0 R, Armonk, NY, United States). The normality of the variables was established by the Kolmogorov–Smirnov test, and all variables had a normal distribution. The means and standard errors (SEs) were calculated for all variables. To determine differences in the measured variables over time, a paired-sample Student's t-test was performed between the two groups. To evaluate the intergroup differences, repeated-measures analysis of variance (ANOVA) was used. For variables measured, an independent Student's t-test and one-way ANOVA followed by the least significant difference (LSD) post-hoc test were performed. The level of significance was determined as a 95% confidence level, with P < 0.05.

Results

Changes of blood routine parameters in rats of each group

Routine blood tests in rats showed that the content of RBC, HGB, WBC, Lymph, PLT, Mon, Gran, and MCHC in the high altitude group was all higher than that in the low altitude group. The RBC count and HGB content showed an increasing trend with the increase of hypoxia time(P < 0.05) [Figure 1]. The increase in RBC count and HGB content is an adaptive and compensatory change of the body under the simulated high-altitude conditions. It also indicates that the model was successful.

Figure 1: (a) The histograms of red blood cell count. (b) The histograms of hemoglobin content. (c) The histograms of white blood cell count. (d) The histograms of lymphocyte count. (e) The histograms of platelet count. (f) The histograms of monocyte count. (g) The histograms of granulocyte count. (h) The histograms of mean corpuscular hemoglobin concentration. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05

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Expression analysis of PP4C gene in different tissues at different hypoxia time

After reverse transcription of RNAs from rat heart, lung, and brain tissue as templates, the cDNA template as well as designed β-actin and PP4 primers were used to quantify mRNA level of PP4 through qPCR. The agarose gel electrophoresis results showed that the amplified product fragments were consistent with the target fragment sizes (69 bp and 143 bp for β-actin and PP4C, respectively), and the amplified products were also confirmed via DNA sequencing [Figure 2].

Figure 2: Agarose gel electrophoresis detection of amplified products of β-actin and PP4 (M: DNA molecular weight marker 1: β-actin 2: PP4C)

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Quantitative results showed that the relative expression levels of PP4C in the heart, brain, and lung tissues of rats were different with different hypoxia times at the simulated high altitude of 4,500 m, and the highest expression levels were formed when entering high altitude area slowly (20-2200 m-7 d-4500 m-3 d). The simulated high altitude hypoxia induced an increase in their expression levels. In addition, the expression level of PP4C in the lung tissue was higher than that in the heart and brain tissue at high altitudes (P < 0.05) [Figure 3].

Figure 3: (a) Relative expression levels of PP4C gene in rat heart tissue. (b) Relative expression levels of PP4C gene in rat brain tissue. (c) Relative expression levels of PP4C gene in lung tissue. (d) Relative expression levels of PP4C gene in the heart, lung, and brain tissues at 20 m-2200 m-7 d-4500 m-3 d. ****P < 0.0001, ***P < 0.001, *P < 0.05

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Expression analysis of PP4 protein in different tissues at different hypoxia time

The PPP4C-PPP4R2-PPP4R3A PP4 complex specifically dephosphorylates H2AFX phosphorylated on Ser-140 (gamma-H2AFX) generated during DNA replication and required for DNA double-strand break repair. PP4 dephosphorylates NDEL1 at CDK1 phosphorylation sites and negatively regulates CDK1 activity in interphase by similarity. In response to DNA damage, PP4 catalyzes RPA2 dephosphorylation, an essential step for DNA repair since it allows the efficient RPA2-mediated recruitment of RAD51 to chromatin. Studies have shown that the improvement of the PP4 effect is primarily due to the increase in PP4R2, which enhances the association of phospho-IKKα/βS176/180 with PP4C, leading to dephosphorylation of phospho-IKKα/βS176/180.^[15] Western blot results showed that PP4 was expressed in the heart, lung, and brain tissues of rats [Figure 4], and the expression level of the high-altitude group was higher than that of the plain group (P < 0.05) [Figure 5].

Figure 4: Expression of PP4R2 (protein phosphatase 4, regulatory subunit 2) and PPX (protein phosphatase 4, catalytic subunit) protein in rat heart tissue, rat brain tissue, and rat lung tissue in each group

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Figure 5: Quantitative analysis gray value of PP4R2 and PPX protein levels in rat heart tissue (a and d), rat brain tissue (b, e), and rat lung tissue (c, f), ****P < 0.0001, ***P < 0.001, *P < 0.05

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Discussion

This study found that PP4 was expressed in the heart, lung, and brain tissues of rats, suggesting that PP4 expression in rat tissues might be as ubiquitous as that in human tissues shown in the human protein atlas database (https://www.proteinatlas.org/). The highest PP4C mRNA level was observed in the lung among the three detected tissues of normal rats, which is consistent with the research results of Hu.^[16] This may be due to the fact that the environment of low pressure and hypoxia at high altitude mainly affects the cardiopulmonary function of the human body. At a high altitude of 4,500 m, the RBC count and HGB content increased, and significant changes in both PP4 mRNA and protein levels were observed in the brain, lung, and heart tissues of rats.^[17] Compared with low altitude groups, PP4 expression in rats was significantly increased at high altitudes (P < 0.05). It may have something to do with low-pressure and low-oxygen exposure. Acute exposure to the low-pressure and low-oxygen environments can cause damage to cardiomyocytes, while chronic exposure to the low-pressure and low-oxygen environment can increase the number of mitochondria and improve the maximum utilization rate of limited oxygen by increasing mitochondrial biosynthesis. By regulating the activity of metabolism-related kinases, the metabolic substrate conversion can be promoted to increase glucose utilization and reduce fatty acids utilization.^[18]

Excessive hyperplasia of RBC will cause high altitude erythrocytosis, high blood viscosity, local blood microcirculation disorders, and affect tissue perfusion to aggravate hypoxia. The increase of RBC count and HGB content is an adaptive and compensatory change of organism.^[19] The PP4C gene expression level in the lung of 20 m-2200 m-7 d-4500 m-7 d rats was significantly different from that of 20 m-2200 m-7 d-4500 m-3 d rats. And its expression level of other groups was lower than that of 20 m-2200 m-7 d-4500 m-3 d group. This may be because, with the prolongation of hypoxia, the rats gradually adapted to the high-altitude environment and their metabolism in the body tended to be stable.^[20] Overall, the order of PP4 expression level in different tissues at high altitude was lung, brain, heart in turn.^[21],[22] This may be mainly due to the low oxygen content in high-altitude areas, which stimulates the lung most significantly.^{[23],[24],[25]} Another factor is that cardiomyocytes increase the resistance to stress by increasing the expression of anti-apoptotic factors. The adaptation of these cardiomyocytes to chronic hypobaric and hypoxic environments is called the chronic hypoxic response of the myocardium.^[26] It indicates that the PP4 protein responds differently to the cold and hypoxic environment in the lung and brain tissues of rats.

Conclusion

The regulatory subunit PP4R2 and the catalytic subunit PPX of PP4 phosphatase complex are both expressed in the heart, brain, and lung of rats, with the highest expression levels in lung tissue. The simulated high altitude hypoxia could induce an increase in their expression levels. These results indicate that the PP4 phosphatase complex is ubiquitously expressed in rat tissue and is likely involved in the adaptation or disease associated with high-altitude hypoxia.

Acknowledgements

We thank Qinghai Research Key Laboratory for echinococcosis for their cooperation in supplying the rats.

Statement of Ethics

Our study was conducted ethically in accordance with the World Medical Association Declaration of Helsinki and has been approved by the Ethics Committee of Qinghai University Affiliated Hospital on animal research.

Author contributions

The research was designed and conducted by Meiyuan Tian and Jing Hou. Meiyuan Tian did the q-PCR experiment, analyzed the data, and prepared the manuscript; Jing Hou did the western blot experiment. Yanyan Ma revised the English language. The final version of the manuscript has been read and approved by all authors and each author believes that the manuscript represents honest work.

Financial support and sponsorship

This study was financially supported by the Qinghai Science and Technology Foundation (No. 2019-ZJ-922).

Conflicts of interest

There are no conflicts of interest.

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