Phosphoprotein phosphatase 1CB (PPP1CB), a novel adipogenic activator, promotes 3T3-L1 adipogenesis

https://doi.org/10.1016/j.bbrc.2015.10.004Get rights and content

Highlights

  • PPP1CB is elevated in vitro in early adipogenesis and in an obese mouse model.

  • PPP1CB knockdown prevents adipocyte differentiation and clonal expansion.

  • PPP1CB knockdown reduces the expression of adipogenic markers.

  • PPP1CB is a promising therapeutic target for treatment of obesity.

Abstract

Understanding the molecular networks that regulate adipogenesis is crucial for gaining insight into obesity and identifying medicinal targets thereof is necessary for pharmacological interventions. However, the identity and molecular actions of activators that promote the early development of adipocytes are still largely unknown. Here, we demonstrate a novel role for phosphoprotein phosphatase 1CB (PPP1CB) as a potent adipogenic activator that promotes adipocyte differentiation. PPP1CB expression increased in vitro during the early phase of 3T3-L1 adipogenesis and in the murine model of high-fat diet-induced obesity. Depletion of PPP1CB dramatically suppressed the differentiation of 3T3-L1 cells into mature adipocytes, with a concomitant change in adipocyte marker genes and significantly inhibited clonal expansion. We also showed that knockdown of PPP1CB caused a significant decrease in C/EBPδ expression, which in turn resulted in attenuation of PPARγ, C/EBPα, adiponectin, and aP2. In addition, we elucidated the functional significance of PPP1CB by linking p38 activation to C/EBPδ expression in early adipogenesis. Overall, our findings demonstrate a novel function of PPP1CB in promoting adipogenesis and suggest that PPP1CB may be a promising therapeutic target for treatment of obesity and obesity-related diseases.

Introduction

Protein phosphatases are important enzymes in diverse eukaryotic physiological processes including cell proliferation, differentiation, cell–cell communication, regulation of transmembrane, and intracellular signaling pathways. Deregulation of their expression and/or activity has been associated with a number of human diseases, including cancer, diabetes, obesity, and autoimmune dysfunction. They can be divided into serine/threonine phosphatases, tyrosine phosphatases, and dual-specific phosphatases that cleave both serine/threonine and tyrosine phosphate groups. Notably, the phosphorylation of cellular proteins in eukaryotes mainly occurs on serine and threonine residues.

Phosphoprotein phosphatase 1 (PPP1), a major type 1 serine/threonine phosphatase, is ubiquitously expressed and modulates a variety of cellular functions including metabolism, cell division, muscle contractility [1]. PPP1 exists as a holoenzyme composed of a catalytic subunit (PPP1C) and a regulatory subunit (PPP1R or PPP1 interacting protein, PIP). In mammals, PPP1C is encoded by three separate genes (PPP1CA, PPP1CB, and PPP1CC); alternative splicing within each generates a variety of PPP1 holoenzymes and results in diverse functionalities. PPP1C isoforms are differentially expressed, depending on the cell type or tissue and even cellular localization, but they possess similar functional properties in vitro [2], [3]. Analysis of a neighbor-joining phylogenetic tree based on alignment of the amino acid sequences reveals that PPP1CC isoforms are more closely related to PPP1CA than PPP1CB [1]. Collectively, these features of PPP1C isoforms reflect that the isoforms can serve distinct—but sometimes overlapping—physiological functions in a context-dependent manner.

Many studies have demonstrated the diverse roles of PPP1C isoforms in regulating cellular functions. PPP1CA has been extensively studied for its role in the cell cycle and apoptosis in immune cells, inducing cell cycle arrest and apoptosis through its Bcl-2 family substrates [4]. It is also closely involved in centrosome-related functions and protein synthesis [5]. The isoform PPP1CB is muscle-specific and involved in glycogen metabolism and muscle contraction [6], [7]. Recently, a role for PPP1CB in cardiomyocytes was also shown: it is the myosin light chain phosphatase responsible for Ca2+-transient rise and increased cell shortening [8]. Finally, the role of PPP1CC1 in regulation of mitosis and metabotropic glutamate receptor internalization was recently highlighted [9]. However, the expression patterns and roles of PPP1C isoforms in adipocytes are poorly understood.

Obesity is a global epidemic as well as a major risk factor for diabetes, hypertension, and cardiovascular diseases [10]. It is mainly caused by excess adipose tissue, which arises from an imbalance between energy intake and expenditure. The major feature of obesity is excess storage of lipids in adipocytes; large adipocytes then secrete more free fatty acids. Generally, preadipocytes and progenitor cells, both of which reside in adipose tissue, undergo a process of terminal differentiation into mature adipocytes. Therefore, identification of novel regulators for controlling the process of adipogenesis and elucidation of their underlying molecular mechanisms is necessary for development of new therapeutic strategies to treat obesity and obesity-related diseases.

Adipogenesis is a well-orchestrated, multistep process coordinated by the interplay of numerous transcription factors, including those in the CCAAT/enhancer-binding family, such as C/EBPα, C/EBPβ, C/EBPδ, and peroxisome proliferator-activated receptor γ (PPARγ) [11]. Among these, C/EBPβ and C/EBPδ expression are some of the earliest events in adipogenesis [12]. Induction of C/EBPβ and C/EBPδ stimulates the expression of C/EBPα and PPARγ, which, in turn, activate transcription of adipogenic genes [13]. Therefore, induction of C/EBPβ and C/EBPδ is essential for the progression of adipogenesis from its earliest stage. Meanwhile, the sequential transcriptional activation of C/EBPβ and C/EBPδ is required for the activation of many MAPK-related signal transduction cascades such as extracellular signal-regulated kinase (ERK), p38, JNK, and glycogen synthase kinase 3β (GSK-3β) [14]. Recently, C/EBPβ and C/EBPδ regulation of early adipocyte differentiation through the involvement of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway genes was also described [15].

In the present study, we investigated the expression of PPP1C isoforms during adipogenesis and their role in controlling adipogenesis. We also elucidated the underlying mechanism of PPP1C isoform-mediated adipogenesis, demonstrating for the first time that PPP1CB is essential for adipocyte differentiation. This study provides novel mechanistic insights into PPP1CB regulation of the early adipogenic process.

Section snippets

Chemicals and antibodies

Dexamethasone, isobutylmethylxanthine (IBMX), insulin, Oil Red O powder, and PPP1CB antibody were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies specific to polyclonal peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins (C/EBPs), adiponectin, fatty acid-binding protein 4 (FABP4/aP2), p-ERK, ERK, p-p38, p38, p-JNK, and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). The β-actin antibody was purchased from Abcam (Cambridge,

Expression of PPP1C isoforms during adipogenesis

To clarify the involvement of PPP1C in adipocyte differentiation, we first examined the expression pattern of the PPP1C isoforms, PPP1CA, PPP1CB, PPP1CC, during differentiation of 3T3-L1 preadipocytes into mature fat cells, which are a well-characterized in vitro model of adipocyte differentiation. Interestingly, PPP1CB increased in a time-dependent manner during the early phase of adipogenesis, as shown by RT-PCR (Fig. 1A) and western blot analysis (Fig. 1B). In contrast, no significant change

Discussion

The differentiation of preadipocytes into mature fat cells is regulated by a highly orchestrated gene expression program. Although many adipogenesis-controlling transcriptional factors have been proposed, the detailed molecular mechanisms of their expression are not fully elucidated. Our study has shown for the first time that PPP1CB is a novel regulator of adipocyte differentiation that functions by regulating C/EBPδ expression during the differentiation of 3T3-L1 preadipocytes. Notably, after

Acknowledgments

This study was supported by the Bio-Synergy Research Project (NRF-2012M3A9C4048775, NRF-2013M3A9C4078151, and NRF-2014M3A9C4066458) through the National Research Foundation of Korea, which was funded by the Ministry of Science, ICT, and Future Planning.

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These authors contributed equally to the work.

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