PPM1A Controls Diabetic Gene Programming through Directly Dephosphorylating PPARγ at Ser273

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipose tissue biology. In obesity, phosphorylation of PPARγ at Ser273 (pSer273) by cyclin-dependent kinase 5 (CDK5)/extracellular signal-regulated kinase (ERK) orchestrates diabetic gene reprogramming via dysregulation of specific gene expression. Although many recent studies have focused on the development of non-classical agonist drugs that inhibit the phosphorylation of PPARγ at Ser273, the molecular mechanism of PPARγ dephosphorylation at Ser273 is not well characterized. Here, we report that protein phosphatase Mg2+/Mn2+-dependent 1A (PPM1A) is a novel PPARγ phosphatase that directly dephosphorylates Ser273 and restores diabetic gene expression which is dysregulated by pSer273. The expression of PPM1A significantly decreases in two models of insulin resistance: diet-induced obese (DIO) mice and db/db mice, in which it negatively correlates with pSer273. Transcriptomic analysis using microarray and genotype-tissue expression (GTEx) data in humans shows positive correlations between PPM1A and most of the genes that are dysregulated by pSer273. These findings suggest that PPM1A dephosphorylates PPARγ at Ser273 and represents a potential target for the treatment of obesity-linked metabolic disorders.


Introduction
Obesity, defined as an accumulation of excess body fat, is closely associated with metabolic diseases, such as dyslipidemia, type 2 diabetes, cardiovascular diseases, and certain types of cancer [1][2][3][4]. Adipose tissue plays a pivotal role in excess energy storage and is considered an important regulator of glucose and lipid metabolism, mediated through the secretion of adipokines [5][6][7]. Obesity is characterized by abnormal adipose tissue and the dysregulation of adipokine secretion [8][9][10]. For example, the expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and chemokines, such as chemokine ligand 2 (CCL2), are higher in obese adipose tissue, whereas the secretion of the insulin-sensitizing adipokines, adiponectin and adipsin, are lower in those tissues [11][12][13][14][15][16][17]. These circulating adipokines specifically orchestrate systemic metabolism by regulating metabolism in peripheral tissues, such as liver, muscle, and macrophages. Thus, an understanding of adipose tissue biology is critical for the most effective treatment of obesity and metabolic diseases.
To check the effect of catalytic inactive mutants of PPM1A (R174G and D239N) in dephosphorylation of PPARγ, HEK-293 cells were transfected with mutants expressing vector. Immunopurified PPM1As were incubated with phosphorylated GST-fused PPARγ in PPM1A phosphatase buffer for 30 min at 30 • C. All reactions were terminated by the addition of protein sample buffer. Phosphorylation of PPARγ was analyzed by SDS-PAGE with phospho-specific antibody against PPARγ at Ser273, anti-GST, or PPM1A antibodies.

Dot Blot Assay
PPM1A human recombinant was incubated with phospho-Ser273 peptide, which conserves the amino acid sequence of PPARγ (TGKTTDK-pS-PFVIYDM, New England peptide, Gardner, MA, USA) in PPM1A phosphatase assay buffer for 20 min at 30 • C. A portion was dotted on nitrocellulose membrane and dried for 3 h at real-time (RT). After dry step, the membrane was washed by Tris-buffered saline and Tween 20 (TBST) 3 times per 10 min. Washed membrane was blocked by 5% bovine serum albumin (BSA) dissolved in TBST and immunoblotting was performed with phospho-specific antibody against PPARγ at Ser273 as described previously.

Subcellular Fractionation
Fully differentiated 3T3-L1 cells were washed by ice-cold PBS buffer, and the centrifuged pellet was resuspended with 20 mM hydroxyethyl piperazine ethane sulfonicacid (HEPES) pH 7.4. Using a 1 mL syringe, cells were lysed 10 times by needle suspension. After incubating for 20 min on ice, lysates were fractionated into nuclei in pellet and cytoplasm in supernatant by 720× g centrifugation for 5 min at 4 • C. Then the pellet was resuspended with 5 mM HEPES pH 7.9 added with 400 mM NaCl and needle suspension was performed 20 times. After 30 min on ice, nuclear proteins acquired by centrifugation at 24,000× g for 30 min at 4 • C. Cytoplasmic and nuclear proteins were quantified by Bradford assay and proteins were detected by Western blotting.

In Vitro Insulin-Resistance (IR) Model
For an experimental procedure of IR-induced model in vitro, we followed Lo. et. al. [39]. Briefly, fully differentiated 3T3-L1 cells were washed by PBS buffer, and changed to low-glucose (1 g/L) DMEM containing 0.5% BSA and 2.5 nM of TNF-α, without serum. After 24 h, total RNAs were isolated.

Real-Time RT-PCR (Quantitative PCR, qPCR)
Total RNAs were isolated using TRIzol reagent purchased from Thermo Fisher Scientific (Waltham, MA, USA). Reverse-transcription of the RNA was performed with ABI Reverse Transcription Kit. qPCR was performed using 7900HT Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Relative mRNA expression levels of each gene were normalized to 36B4 or TATA-binding protein TBP. Each analysis was conducted in triplicates. Data were analyzed by Microsoft Excel (Microsoft, Redmond, WA, USA).

Correlation Analysis in Human Adipose Tissues
To analyze the correlation between PPM1A and PPARγ phosphorylation at Ser273 in lean and obese humans, GSE55200, from public Gene Expression Omnibus (GEO) database accessible at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), was analyzed. We analyzed the expression levels of PPM1A and genes responsive to PPARγ phosphorylation at Ser273 as described previously in human subcutaneous adipose tissue [40,41].

Animals
All animal experiments were performed according to procedures approved by the Ulsan National Institute of Science and Technology's Institutional Animal Care and Use Committee. Five-week-old male C57BL/6J mice (DBL, Chungbuk, Korea) were fed a high-fat diet (60% kcal fat, D12492, Research Diets Inc., New Brunswick, NJ, USA) for 8 weeks. The db/db mice used in this study were purchased from the Jackson Laboratory (Bar Harbor, ME, USA).

PPM1A is a Phosphatase that Acts on PPARγ
Recent studies showed that members of the PPM family could be the serine phosphatases of PPARγ [31,32]. Therefore, we aimed to identify the phosphatase for the Ser273 residue of PPARγ by interrogating a number of PPMs. Of the seven tested, we found that both PPM1A and PPM1B specifically dephosphorylated PPARγ at Ser273 after phorbol myristate acetate (PMA) treatment, and that PPM1A did so more effectively than PPM1B ( Figure 1A). Consistent with previous reports, PPM1B dephosphorylates PPARγ at Ser112 [31], but interestingly, PPM1A also inhibits PMA-mediated PPARγ phosphorylation at Ser112. To further investigate the effects of PPM1A on the phosphorylation of PPARγ, we overexpressed PPM1A in HEK-293 cells and found that the degree of dephosphorylation of PPARγ by PPM1A is proportional to the level of PPM1A expression ( Figure 1B). It was reported that the phosphorylation of PPARγ at Ser273 is mediated by ERK [29]. Therefore, we tested whether the dephosphorylation by PPM1A might be the result of an inhibition of ERK activation following PMA treatment. However, forced expression of PPM1A did not change the degree of PMA-induced ERK phosphorylation, suggesting that the dephosphorylation of PPARγ by PPM1A is not caused by the indirect inhibition of ERK. Recent studies showed that members of the PPM family could be the serine phosphatases of PPARγ [31,32]. Therefore, we aimed to identify the phosphatase for the Ser273 residue of PPARγ by interrogating a number of PPMs. Of the seven tested, we found that both PPM1A and PPM1B specifically dephosphorylated PPARγ at Ser273 after phorbol myristate acetate (PMA) treatment, and that PPM1A did so more effectively than PPM1B ( Figure 1A). Consistent with previous reports, PPM1B dephosphorylates PPARγ at Ser112 [31], but interestingly, PPM1A also inhibits PMAmediated PPARγ phosphorylation at Ser112. To further investigate the effects of PPM1A on the phosphorylation of PPARγ, we overexpressed PPM1A in HEK-293 cells and found that the degree of dephosphorylation of PPARγ by PPM1A is proportional to the level of PPM1A expression ( Figure  1B). It was reported that the phosphorylation of PPARγ at Ser273 is mediated by ERK [29]. Therefore, we tested whether the dephosphorylation by PPM1A might be the result of an inhibition of ERK activation following PMA treatment. However, forced expression of PPM1A did not change the degree of PMA-induced ERK phosphorylation, suggesting that the dephosphorylation of PPARγ by PPM1A is not caused by the indirect inhibition of ERK.
To determine whether PPM1A phosphatase activity is required for PPARγ dephosphorylation, we generated two catalytically inactive PPM1A mutants, R174G and D239N [34], and found that both lost their ability to dephosphorylate PPARγ at Ser112 and Ser273, while not affecting PMA-induced ERK activation ( Figure 1C). Taken together, these results strongly suggest that PPM1A is a specific phosphatase of PPARγ at both Ser112 and Ser273 in the cell culture system.  To determine whether PPM1A phosphatase activity is required for PPARγ dephosphorylation, we generated two catalytically inactive PPM1A mutants, R174G and D239N [34], and found that both Cells 2020, 9, 343 6 of 18 lost their ability to dephosphorylate PPARγ at Ser112 and Ser273, while not affecting PMA-induced ERK activation ( Figure 1C). Taken together, these results strongly suggest that PPM1A is a specific phosphatase of PPARγ at both Ser112 and Ser273 in the cell culture system.

PPM1A Directly Dephosphorylates PPARγ at Ser273 but not Ser112
We next determined the effect of PPM1A on PPARγ dephosphorylation in a cell-free system. After purifying a glutathione S-transferase (GST)-PPARγ recombinant fusion protein, we directly phosphorylated PPARγ at Ser273 using active ERK. Then, we performed an in vitro phosphatase assay using recombinant active PPM1A. As shown in Figure 2A, PPM1A directly dephosphorylated PPARγ at Ser273 but not at Ser112. These results indicate that PPM1A is a direct phosphatase of PPARγ at Ser273, and that the dephosphorylation by PPM1A at Ser112 in cells is indirect. Furthermore, when we incubated a phosphopeptide including the pSer273 residue with active PPM1A, phosphorylation at Ser273 significantly decreased ( Figure 2B). Due to PPM1A [33,34], we further tested the effect of both ions on dephosphorylating PPARγ at Ser273. As shown in Figure 2C,D, the phosphatase activity of PPM1A against pSer273 is much higher in the presence of both Mg 2+ and Mn 2+ ions, but pSer112 is unchanged. Next, to determine whether the catalytic activity of PPM1A is required for the dephosphorylation of PPARγ, we immunopurified wild-type (WT) PPM1A and the two catalytic inactive mutants of PPM1A from HEK-293 cells. After phosphorylating PPARγ at Ser273, we incubated phosphorylated PPARγ with PPM1As. WT PPM1A, but not the two catalytically inactive mutants, dephosphorylated PPARγ ( Figure 2E). Taken together, these results strongly suggest that PPM1A is a specific and direct phosphatase of PPARγ at Ser273, but not Ser112.

PPM1A Directly Dephosphorylates PPARγ at Ser273 but not Ser112
We next determined the effect of PPM1A on PPARγ dephosphorylation in a cell-free system. After purifying a glutathione S-transferase (GST)-PPARγ recombinant fusion protein, we directly phosphorylated PPARγ at Ser273 using active ERK. Then, we performed an in vitro phosphatase assay using recombinant active PPM1A. As shown in Figure 2A, PPM1A directly dephosphorylated PPARγ at Ser273 but not at Ser112. These results indicate that PPM1A is a direct phosphatase of PPARγ at Ser273, and that the dephosphorylation by PPM1A at Ser112 in cells is indirect. Furthermore, when we incubated a phosphopeptide including the pSer273 residue with active PPM1A, phosphorylation at Ser273 significantly decreased ( Figure 2B). Due to PPM1A [33,34], we further tested the effect of both ions on dephosphorylating PPARγ at Ser273. As shown in Figure  2C,D, the phosphatase activity of PPM1A against pSer273 is much higher in the presence of both Mg 2+ and Mn 2+ ions, but pSer112 is unchanged. Next, to determine whether the catalytic activity of PPM1A is required for the dephosphorylation of PPARγ, we immunopurified wild-type (WT) PPM1A and the two catalytic inactive mutants of PPM1A from HEK-293 cells. After phosphorylating PPARγ at Ser273, we incubated phosphorylated PPARγ with PPM1As. WT PPM1A, but not the two catalytically inactive mutants, dephosphorylated PPARγ ( Figure 2E). Taken together, these results strongly suggest that PPM1A is a specific and direct phosphatase of PPARγ at Ser273, but not Ser112.

PPM1A Physically Interacts with PPARγ
Since PPM1A directly dephosphorylates PPARγ, it is expected that they would be localized to the nucleus. Thus, we analyzed the subcellular localization of both PPM1A and PPARγ and found that both were predominantly localized in the nucleus ( Figure 3A). Next, we performed co-immunoprecipitation experiments to determine whether PPM1A binds to PPARγ. Endogenous PPM1A specifically interacts with endogenous PPARγ in adipocytes and vice versa ( Figure 3B,C). In addition, PPM1A specifically interacts with PPARγ when co-expressed in HEK-293 cells ( Figure 3D). Interestingly, a phosphorylation-defective mutant of PPARγ (S273A) interacts normally with PPM1A. Furthermore, PMA does not affect the interaction between PPM1A and PPARγ, suggesting that the phosphorylation status of PPARγ is not critical for its interaction with PPM1A. The catalytically inactive PPM1A mutant (R174G) retains its ability to interact with PPARγ, but the D239N mutation significantly reduces the interaction ( Figure 3F). These results are consistent with previous reports that Asp239 residue is critical for substrate binding by coordinating to metal M2 and bridging with metal-bound water [42]. Together, these results indicate that PPM1A physically interacts with PPARγ in a phosphorylation-independent manner. Since PPM1A directly dephosphorylates PPARγ, it is expected that they would be localized to the nucleus. Thus, we analyzed the subcellular localization of both PPM1A and PPARγ and found that both were predominantly localized in the nucleus ( Figure 3A). Next, we performed coimmunoprecipitation experiments to determine whether PPM1A binds to PPARγ. Endogenous PPM1A specifically interacts with endogenous PPARγ in adipocytes and vice versa ( Figure 3B,C). In addition, PPM1A specifically interacts with PPARγ when co-expressed in HEK-293 cells ( Figure 3D). Interestingly, a phosphorylation-defective mutant of PPARγ (S273A) interacts normally with PPM1A. Furthermore, PMA does not affect the interaction between PPM1A and PPARγ, suggesting that the phosphorylation status of PPARγ is not critical for its interaction with PPM1A. The catalytically inactive PPM1A mutant (R174G) retains its ability to interact with PPARγ, but the D239N mutation significantly reduces the interaction ( Figure 3F). These results are consistent with previous reports that Asp239 residue is critical for substrate binding by coordinating to metal M2 and bridging with metal-bound water [42]. Together, these results indicate that PPM1A physically interacts with PPARγ in a phosphorylation-independent manner.

PPM1A Specifically Regulates the Expression of a Diabetic Gene Set that is Dysregulated by pSer273
To elucidate the functional roles of PPM1A on pSer273, we investigated the effect of PPM1A on pSer273-mediated gene regulation in adipocytes. Specific siRNAs targeting PPM1A were introduced into adipocytes, which dramatically reduce the expression of PPM1A ( Figure 4A). The expression of PPARγ is not changed by PPM1A knockdown ( Figure 4B). Importantly, most of the genes known to

PPM1A Specifically Regulates the Expression of a Diabetic Gene Set that is Dysregulated by pSer273
To elucidate the functional roles of PPM1A on pSer273, we investigated the effect of PPM1A on pSer273-mediated gene regulation in adipocytes. Specific siRNAs targeting PPM1A were introduced into adipocytes, which dramatically reduce the expression of PPM1A ( Figure 4A). The expression of PPARγ is not changed by PPM1A knockdown ( Figure 4B). Importantly, most of the genes known to be specifically dysregulated by pSer273 are sensitive to the knockdown of PPM1A ( Figure 4C). These include adiponectin and adipsin, which are adipokines that are important regulators of insulin sensitivity and glucose homeostasis [14][15][16][17]. 8 be specifically dysregulated by pSer273 are sensitive to the knockdown of PPM1A ( Figure 4C). These include adiponectin and adipsin, which are adipokines that are important regulators of insulin sensitivity and glucose homeostasis [14][15][16][17].
In contrast, the forced expression of PPM1A in adipocytes has opposite effects to the knockdown of PPM1A with gene regulation. The lentiviral overexpression of WT PPM1A and two catalytic inactive mutants does not change the expression of PPARγ in adipocytes ( Figure 5A,B). However, WT PPM1A markedly increases the expression of most of the specific gene set that is dysregulated by pSer273, but the R174G and D239N mutants do not ( Figure 5C). Taken together, these results strongly suggest that PPM1A regulates the diabetic gene program by dephosphorylating PPARγ at Ser273.  In contrast, the forced expression of PPM1A in adipocytes has opposite effects to the knockdown of PPM1A with gene regulation. The lentiviral overexpression of WT PPM1A and two catalytic inactive mutants does not change the expression of PPARγ in adipocytes ( Figure 5A,B). However, WT PPM1A markedly increases the expression of most of the specific gene set that is dysregulated by pSer273, but the R174G and D239N mutants do not ( Figure 5C). Taken together, these results strongly suggest that PPM1A regulates the diabetic gene program by dephosphorylating PPARγ at Ser273.

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Western blotting and real-time PCR analysis revealed that PPM1A is expressed in pre-adipocytes, and that its expression significantly increases during adipogenesis ( Figure 6A). In addition, the expression of PPARγ and adipogenic marker genes, including aP2, C/EBPα, and adiponectin, also significantly increases ( Figure 6B), indicating that PPM1A might have important roles in differentiated adipocytes. It is well characterized that pS273 is linked to obesity and insulin resistance [28,29] and blocking pSer273 by specific PPARγ ligands shows improved insulin sensitivity [43][44][45].

Knockdown of PPM1A Devastates Insulin Resistant Status in Adipocytes
Next, we aimed to determine the physiological relevance of PPM1A in adipocyte biology. Western blotting and real-time PCR analysis revealed that PPM1A is expressed in pre-adipocytes, and that its expression significantly increases during adipogenesis ( Figure 6A). In addition, the expression of PPARγ and adipogenic marker genes, including aP2, C/EBPα, and adiponectin, also significantly increases ( Figure 6B), indicating that PPM1A might have important roles in differentiated adipocytes. It is well characterized that pS273 is linked to obesity and insulin resistance [28,29] and blocking pSer273 by specific PPARγ ligands shows improved insulin sensitivity [43][44][45].
Thus, we tried to determine whether PPM1A has a potential role in insulin sensitivity. It was shown that insulin resistance can be modeled by treating fully differentiated adipocytes with TNF-α [39]. Therefore, we treated adipocytes with TNF-α, which dramatically increased the expression of genes involved in insulin resistance in adipocytes, such as IL-6, CCL2, and chemokine ligand 9 (CCL9), but significantly reduced glucose transporter 4 (GLUT4) expression ( Figure 6C). Interestingly, the expression of PPM1A was significantly decreased in this model, and those genes were dramatically altered by PPM1A knockdown in adipocytes. The expression of IL-6, CCL2, and CCL9 dramatically increases by specific knockdown of PPM1A and the expression of GLUT4 is significantly decreased by PPM1A knockdown. These results strongly suggest that PPM1A is positively associated with insulin sensitivity in adipocytes. Thus, we tried to determine whether PPM1A has a potential role in insulin sensitivity. It was shown that insulin resistance can be modeled by treating fully differentiated adipocytes with TNFα [39]. Therefore, we treated adipocytes with TNF-α, which dramatically increased the expression of genes involved in insulin resistance in adipocytes, such as IL-6, CCL2, and chemokine ligand 9 (CCL9), but significantly reduced glucose transporter 4 (GLUT4) expression ( Figure 6C). Interestingly, the expression of PPM1A was significantly decreased in this model, and those genes were dramatically altered by PPM1A knockdown in adipocytes. The expression of IL-6, CCL2, and CCL9 dramatically increases by specific knockdown of PPM1A and the expression of GLUT4 is significantly decreased by PPM1A knockdown. These results strongly suggest that PPM1A is positively associated with insulin sensitivity in adipocytes.

PPM1A is Negatively Correlated with pSer273
We then investigated the physiological relevance of PPM1A in adipose tissue. First, we used two different models: high-fat diet (HFD)-induced obese mice and genetically obesity mice (db/db). As shown in Figure 7A,B, the phosphorylation of PPARγ at Ser273 is very high in the adipose of these mice, as previously reported [28]. Interestingly, PPM1A expression was significantly lower in the adipose tissue of both HFD-fed and db/db mice than in control mice, indicating that there is a negative association between PPM1A expression and pSer273 ( Figure 7C,D). To further evaluate this correlation, we interrogated a public GSE55200 database which compares gene expression in lean and obese human subcutaneous adipose tissue ( Table 1). The expression of PPM1A is significantly lower in obese than in normal adipose. By contrast, the expression of pro-inflammatory cytokines,

PPM1A is Negatively Correlated with pSer273
We then investigated the physiological relevance of PPM1A in adipose tissue. First, we used two different models: high-fat diet (HFD)-induced obese mice and genetically obesity mice (db/db). As shown in Figure 7A,B, the phosphorylation of PPARγ at Ser273 is very high in the adipose of these mice, as previously reported [28]. Interestingly, PPM1A expression was significantly lower in the adipose tissue of both HFD-fed and db/db mice than in control mice, indicating that there is a negative association between PPM1A expression and pSer273 ( Figure 7C,D). To further evaluate this correlation, we interrogated a public GSE55200 database which compares gene expression in lean and obese human subcutaneous adipose tissue ( Table 1). The expression of PPM1A is significantly lower in obese than in normal adipose. By contrast, the expression of pro-inflammatory cytokines, such as TNF-α and CCL2, is much higher. Furthermore, the expression of adiponectin and GLUT4 is significantly lower. In addition, most of the genes dysregulated by pSer273 are significantly increased in obese adipose tissue (Table 1). To further check the correlation between PPM1A expression and phosphorylation of PPARγ at Ser273, we analyzed the relative expression of PPM1A and pSer273 specific genes in human subcutaneous adipose tissue using GTEx database ( Figure 7E). Indeed, the expression of most of the genes dysregulated by pSer273 positively correlated with the expression of PPM1A. Taken together, our results strongly suggest that PPM1A is negatively correlated with pSer273 in both mice and humans. such as TNF-α and CCL2, is much higher. Furthermore, the expression of adiponectin and GLUT4 is significantly lower. In addition, most of the genes dysregulated by pSer273 are significantly increased in obese adipose tissue (Table 1). To further check the correlation between PPM1A expression and phosphorylation of PPARγ at Ser273, we analyzed the relative expression of PPM1A and pSer273 specific genes in human subcutaneous adipose tissue using GTEx database ( Figure 7E). Indeed, the expression of most of the genes dysregulated by pSer273 positively correlated with the expression of PPM1A. Taken together, our results strongly suggest that PPM1A is negatively correlated with pSer273 in both mice and humans.  Expression of PPM1A, pSer273 responsive genes (Cd24a, Txnip, Nr1d1, Aplp2, Peg10, Acyl, Cidec, Nr3c1, Rarres2, Car3, Selenbp1, Nr1d2, Ddx17, Rybp, Adiponectin, and Adipsin), insulin sensitivity-related genes (Glut4), and insulin resistance-related genes (Tnf, Ccl2) in human adipose tissue is analyzed (GSE55200). Log fold changes (log 2 FC) of unhealthy obese individuals (n = 8) versus lean healthy individuals (n = 7) are calculated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Discussion
Post-translational modifications of PPARγ orchestrate various functions of the protein [18,25,26]. In obesity, phosphorylation of PPARγ on the Ser273 residue by CDK5 leads to dysregulation of a specific gene set [28]. In addition to the effect of CDK5 on Ser273, ERK is shown to regulate the diabetogenic effect of PPARγ [29]. These studies collectively suggest that pSer273 is a potential therapeutic target for metabolic diseases. In this study, we demonstrate that PPM1A is a novel protein phosphatase of PPARγ that directly dephosphorylates pSer273, reprogramming anti-diabetic gene expression (Figure 8). First, PPM1A interacts with PPARγ and directly dephosphorylates at Ser273. Second, genetically altered expression of PPM1A significantly affects the expression of the gene set that is dysregulated by pSer273 in adipocytes. Third, PPM1A expression negatively correlates with the degree of phosphorylation of PPARγ, both in obese mice and human adipose tissue. Finally, transcriptomic analyses are highly consistent with this relationship.

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phosphatase of PPARγ that directly dephosphorylates pSer273, reprogramming anti-diabetic gene expression (Figure 8). First, PPM1A interacts with PPARγ and directly dephosphorylates at Ser273. Second, genetically altered expression of PPM1A significantly affects the expression of the gene set that is dysregulated by pSer273 in adipocytes. Third, PPM1A expression negatively correlates with the degree of phosphorylation of PPARγ, both in obese mice and human adipose tissue. Finally, transcriptomic analyses are highly consistent with this relationship. Although a number of studies identified phosphatases of PPARγ, most focused on pSer112. Our results show that PPM1A reduces the level of phosphorylation at Ser112, but an in vitro phosphatase assay indicates that this dephosphorylation is not direct. The Ser112 residue of PPARγ is phosphorylated by ERK and c-Jun N-terminal kinase (JNK), but not p38 MAPK, and reduces PPARγ activity. Paradoxically, the same site can be phosphorylated by CDK7 and CDK9, which increases the activity of PPARγ [46,47]. We showed that there is no effect of PPM1A on the phosphorylation of ERK in cells (Figure 1), but the exact mechanism whereby PPM1A reduces pSer112 in adipocytes remains to be elucidated. Further mechanistic studies should be conducted to confirm the direct target of PPM1A that affects phosphorylation at Ser112.
Recent studies have shown that PPM1A plays an important role in metabolism [37,[48][49][50]. Unhealthy adipocytes are exposed to adipose tissue-resident macrophages, which are phenotypically defined as M1 macrophages [2,[51][52][53][54]. Pro-inflammatory cytokines, including IL-1β, IL6, and TNF-α, secreted by M1-polarized macrophages, alter the metabolic phenotype of adipose tissue [55][56][57][58]. Smith et al. showed that PPM1A regulates monocyte-macrophage differentiation, reduces the expression of M1 macrophage marker genes, and inhibits the production of pro-inflammatory cytokines [59]. In addition, PPM1A terminates TNF-α-induced inhibitor κB kinase beta (IKKβ) activation and transforming growth factor beta (TGFβ) signaling by dephosphorylating IKKβ and Although a number of studies identified phosphatases of PPARγ, most focused on pSer112. Our results show that PPM1A reduces the level of phosphorylation at Ser112, but an in vitro phosphatase assay indicates that this dephosphorylation is not direct. The Ser112 residue of PPARγ is phosphorylated by ERK and c-Jun N-terminal kinase (JNK), but not p38 MAPK, and reduces PPARγ activity. Paradoxically, the same site can be phosphorylated by CDK7 and CDK9, which increases the activity of PPARγ [46,47]. We showed that there is no effect of PPM1A on the phosphorylation of ERK in cells (Figure 1), but the exact mechanism whereby PPM1A reduces pSer112 in adipocytes remains to be elucidated. Further mechanistic studies should be conducted to confirm the direct target of PPM1A that affects phosphorylation at Ser112.
Recent studies have shown that PPM1A plays an important role in metabolism [37,[48][49][50]. Unhealthy adipocytes are exposed to adipose tissue-resident macrophages, which are phenotypically defined as M1 macrophages [2,[51][52][53][54]. Pro-inflammatory cytokines, including IL-1β, IL6, and TNF-α, secreted by M1-polarized macrophages, alter the metabolic phenotype of adipose tissue [55][56][57][58]. Smith et al. showed that PPM1A regulates monocyte-macrophage differentiation, reduces the expression of M1 macrophage marker genes, and inhibits the production of pro-inflammatory cytokines [59]. In addition, PPM1A terminates TNF-α-induced inhibitor κB kinase beta (IKKβ) activation and transforming growth factor beta (TGFβ) signaling by dephosphorylating IKKβ and SMAD2/3, respectively [38,60]. These results suggest that PPM1A plays an important role in inhibiting pro-inflammatory cytokine-induced inflammatory signaling. Interestingly, our previous studies demonstrated that dephosphorylation of PPARγ at Ser273 is responsible for anti-inflammatory functions by controlling macrophage polarization which decreased M1 polarization, but increased M2 phenotypes [44,45]. Furthermore, we found that TNF-α-induced phosphorylation of PPARγ at Ser273 increased pro-inflammatory response in adipocytes [45]. Notably, insulin-resistant TNF-α-treated differentiated 3T3-L1 adipocytes show significantly lower PPM1A expression. Specifically, PPM1A recovers insulin sensitivity-related gene expression which is dysregulated by TNF-α ( Figure 6) and upregulates the expression of insulin-sensitizing adipokines, adiponectin, and adipsin (Figures 4 and 5). Together, we highlight that PPM1A may represent a therapeutic target for metabolic diseases in many aspects, such as increasing anti-inflammatory responses and enhancing the secretion of insulin-sensitizing adipokines which depends on dephosphorylation of PPARγ at Ser273. Both extracellular and intracellular magnesium deficits are frequently detected in patients with type 2 diabetes and in overweight or obese children [61,62]. Low Mg 2+ concentrations are closely associated with post-receptor impairment in the insulin signaling pathway [61,63,64]. In addition, a series of recent studies revealed lower concentrations of Mg 2+ in the liver, small intestine, and bone of lean mice compared with obese mice [65][66][67]. Furthermore, epidemiological studies conducted worldwide suggest that a higher intake of Mn 2+ is associated with a lower risk of obesity [61,68,69]. Thus, the Mg 2+ /Mn 2+ -dependency of PPM1A for the dephosphorylation of PPARγ is an important consideration when evaluating the phosphatase as a therapeutic target. However, the intracellular concentrations of Mg 2+ and Mn 2+ are strictly regulated, and failure of homeostasis is involved in several diseases [70,71]. For example, hypermagnesemia is common in renal failure patients [72], and an accumulation of manganese in the brain can cause neurodegenerative disorders [73]. Thus, further studies are required to uncover the molecular mechanisms which promote the expression of PPM1A and to develop the specific activators of PPM1A rather than regulating the intracellular concentrations of Mg 2+ and Mn 2+ .
Our data suggest the critical roles for PPM1A as a novel phosphatase of PPARγ, which finely orchestrates diabetic gene expression in adipocytes. Although further studies are required to elucidate the relationship between PPM1A and metabolic diseases, our study proposes that PPM1A may represent a promising therapeutic target for obesity and metabolic disorders.