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Serpina3c Regulates Adipogenesis by Modulating Insulin Growth Factor 1 and Integrin Signaling

Summary Preadipocyte differentiation can be induced upon a hormonal treatment, and various factors secreted by the cells may contribute to adipogenesis. In this study, RNA-seq revealed Serpina3c as a critical factor regulating the signaling network during adipogenesis. Serpina3c is a secretory protein and is highly expressed in fat tissues. Knockdown of Serpina3c decreased adipogenesis by attenuating the mitotic clonal expansion of 3T3-L1 cells. These cells exhibited decreases in integrin α5, which abolished the phosphorylation of integrin β3. We found that Serpina3c inhibits a serine protease that regulates integrin α5 degradation. Knockdown of Serpina3c disrupted integrin-mediated insulin growth factor 1 (IGF-1) signaling and ERK activation. Serpina3c-mediated regulation of integrin-IGF-1 signaling is also associated with AKT activation, which affects the nuclear translocation of GSK3β. Altogether, our results indicate that Serpina3c secreted from differentiating adipocytes inhibits serine proteases to modulate integrin/IGF-1-mediated ERK and AKT signaling and thus is a critical factor contributing to adipogenesis.


INTRODUCTION
Obesity is a global disease associated with metabolic disorders such as diabetes, hypertension, and cardiovascular disease (Kopelman, 2000). Studies of the complex process by which adipocytes differentiate from preadipocyte precursors (i.e., adipogenesis) are important for understanding the underlying mechanisms of this disease (Gregoire et al., 1998). The 3T3-L1 preadipocyte cell line is an in vitro model widely used to study the molecular mechanisms of adipocyte differentiation (MacDougald and Lane, 1995). When proliferating 3T3-L1 preadipocytes become confluent in culture dishes, they enter G 1 growth-arrest phase (Tang et al., 2003b). Upon treatment with hormonal cocktail (a mixture of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, hereafter called MDI) and fetal bovine serum (FBS), growth-arrested preadipocytes reenter the cell cycle and initiate differentiation via serial expression of adipogenic genes such as those encoding CCAAT/enhancer binding protein (C/EBP) b, C/EBPa, and peroxisome proliferator-activated receptor g (PPARg) (Lin and Lane, 1994;Tang et al., 2003a). This resumed cell cycle is called mitotic clonal expansion, and the process sustains during the first 2-3 days of differentiation and is essential for 3T3-L1 differentiation because an inhibition of this step completely blocks adipogenesis (Tang et al., 2003b).
In our previous studies to identify important differentiation factors secreted by clonally expanding cells, we observed that the differentiation program was accelerated in adipocytes treated with conditioned medium (CM) relative to those treated with MDI . Moreover, the CM-treated cells expressed adipocyte-specific genes before undergoing clonal expansion . Thus, the aim of the present study was to reveal the factors in CM responsible for promoting adipogenesis. To this end, we began by screening gene expression changes in cells treated with CM. We subsequently identified the serine protease Serpina3c as a protein regulating 3T3-L1 preadipocyte differentiation. Serpina3c is a member of the Serpin superfamily, and some Serpin family proteins have been implicated in adipocyte differentiation and obesity in the proteomic study (Zvonic et al., 2007); however, their precise physiological roles are unknown. Serpina3c is classified in the serpin A clade, which comprises 11 genes (SERPINA1 and SERPINA3 to -12) and 2 pseudogenes (SERPINA2 and SERPINA13P) in humans and 9 genes (Serpina3a, -b, -c, and -f to -n) in mice (Heit et al., 2013). Serpina3 is a known inhibitor of chymotrypsin and cathepsin G (Law et al., 2006) and is reported to be expressed in the blood, liver, kidney, and lung (Heit et al., 2013). We reveal here that Serpina3c is highly expressed in adipose tissue of mice and has an important role in adipocyte differentiation. This effect was closely associated with ERK and AKT activation via insulin-like growth factor 1 (IGF-1) signaling, which involves a complex formed by the IGF-1 receptor (IGF-1R) and the integrin a5-b3 heterodimer (Tahimic et al., 2013). We further demonstrate that Serpina3c inhibits a serine protease that degrades integrin a5. Thus, Serpina3c is a critical factor involved in the early signaling pathway of adipogenesis, and inhibition of Serpina3c may offer beneficial effects in the treatment of obesity.

Serpina3c Is Expressed and Secreted by Differentiating Adipocytes
3T3-L1 preadipocytes were cultured to confluence in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Two days later, the cells were treated with MDI and FBS to induce cell-cycle reentry and preadipocyte differentiation. On differentiation day 2, the medium (i.e., CM) was collected and used to treat 3T3-L1 preadipocytes. Cells were then harvested 0, 4, or 24 h after treatment with CM (or MDI as a control) for RNA sequencing ( Figure 1A). As previously observed , the terminal differentiation of CM-treated 3T3-L1 adipocytes was accelerated as evidenced by Oil Red O staining ( Figure 1B). To identify genes associated with this acceleration, we focused on genes (1) upregulated >2-fold by MDI and FBS after 24 h of differentiation, (2) induced immediately (at 4 h of differentiation) by CM, and (3) with a CM/MDI ratio >2-fold at 4 h of differentiation ( Figure 1C). We thought that these criteria would meet a critical condition to find factors that are up-regulated by CM, that is, by the factors secreted by differentiating adipocytes. This enables us to identify~20 candidate genes (Figure 1D; the total gene expression dataset is available in Data S1). Of these, Serpina3c and Serpina3n have attracted immediate attention, because these are predicted as secreting molecules whose roles are not clarified in adipogenesis yet.
We further examined the expression of Serpina3c and Serpina3n during the course of 3T3-L1 differentiation. In cells treated with MDI, Serpina3c expression gradually increased, peaking at 24-48 h, whereas the expression of Serpina3n did not change markedly (Figures 2A and 2B). By contrast, the expression of Serpina3c rapidly reached the highest level soon after CM treatment and maintained this level during the whole differentiation process (Figures 2A and 2B). During the late stage of adipogenesis, the expression levels of adipokines such as leptin and adiponectin were not affected by Serpina3c ( Figure 2C). Serpi-na3c mRNA expression was high in white adipose tissues and liver but much lower in other mouse tissues ( Figure 2D). These data are in accordance with a role for Serpina3c in adipocyte differentiation and the rapid progression of adipogenesis induced by CM.
Serpina3c (also known as alpha 1 proteinase inhibitor antitrypsin [Heit et al., 2013;Law et al., 2006]) is predicted to be a secretory protein with a signal peptide on its N terminus ( Figure 2E). To confirm that it is a secretory protein, we generated Serpina3c and Serpina3n overexpression vectors with FLAG tags and transfected them into HEK293T cells. Serpina3c and -3n proteins were detected in the medium from transfected cells ( Figure 2F), indicating that they are indeed secreted from cells. In addition, the secretion of Serpina3c protein was paralleled with intracellular expression of Serpina3c ( Figure 2G).

Knockdown of Serpina3c Inhibits Mitotic Clonal Expansion and Adipocyte Differentiation
To clarify the role of Serpina3c or Serpina3n in adipogenesis, we knocked down expression via RNA interference. Although Serpina3c and Serpina3n are located on the same chromosome and encode proteins with similar amino acid sequences, only knockdown of Serpina3c affected 3T3-L1 adipogenesis ( Figures  3A and 3B). Interestingly, the effect of Serpina3c knockdown was observed in cells treated with MDI but not with CM, indicating that CM may already contain Serpina3c secreted by differentiating cells. In other words, differentiating 3T3-L1 cells with MDI secrete Serpina3c into the medium (i.e., CM), then CM-treated 3T3-L1 cells no longer require Serpina3c expression.
During mitotic clonal expansion, cell numbers normally increase 2-to 3-fold (Lane et al., 1999;). However, this was not observed in cells with Serpina3c knockdown after MDI induction ( Figure 3C). These results were confirmed by fluorescence-activated cell sorting analysis. The G 1 -S 2 transition was inhibited in cells with Serpina3c knockdown, whereas more control cells reentered the cell cycle after MDI treatment ( Figure 3D). Consistent with a role for Serpina3c in adipogenesis, knockdown of Serpina3c resulted in reduced expression of PPARg and C/EBPa ( Figure 3E). This result suggests that Serpina3c is associated with mitotic clonal expansion in adipogenesis.

Serpina3c Is Involved in Adipogenesis via the Integrin-Mediated IGF-1 Pathway
As human SERPINA3 is reported as an inhibitor of cathepsin G (Heit et al., 2013;Law et al., 2006), a type of serine protease known to modulate integrin clustering in neutrophils and induce integrin cleavage in platelets (Raptis et al., 2005;Si-Tahar et al., 1997), we speculated that integrin regulation may be altered in cells with Serpina3c knockdown. Western blotting revealed that integrin a5 but not integrin a6 was decreased by Serpina3c knockdown ( Figure 4A). Integrin a5 and b3 heterodimerize and interact with IGF-1R (Sekimoto et al., 2005), and in the presence of IGF-1, Tyr759 of integrin b3 is phosphorylated and binds Src (Cowan et al., 2000;Fagerholm et al., 2004). As shown in Figure 4A, integrin b3 phosphorylation was diminished in cells with Serpina3c knockdown, suggesting that integrin-IGF-1R signaling is altered. Accordingly, phosphorylation of the downstream signaling enzyme ERK, which is required for C/EBPb phosphorylation and mitotic clonal expansion in adipogenesis (Kim et al., 2007;Tang et al., 2003a), was decreased by Serpina3c knockdown. Notably, the expression of integrin a5 mRNA was increased by Serpina3c knockdown Figure 1. Gene Expression Profile in Differentiating 3T3-L1 Cells Treated with CM (A) A schematic model of CM preparation using a 3T3-L1 preadipocyte differentiation system. Confluent growth-arrested 3T3-L1 preadipocytes were induced to differentiate with MDI and FBS. After 2 days, cell-exposed medium was collected to treat other preadipocytes. Differentiating cells were harvested after 0, 4, and 24 h for RNA sequencing. MDI + FBStreated cells were used as a control group. (B) CM accelerated 3T3-L1 adipocyte differentiation as shown by Oil Red O staining at day 5 of induction. (C) Strategy for the identification of CM-containing factors via RNA sequencing data. (D) Heatmap showing relative expression of 20 genes identified from RNA sequencing data. Red, high expression; blue, low expression. Fold changes of the total genes are presented in Data S1. These RNA sequencing data using MDI and CM are publicly available from Gene Expression Omnibus (GEO: GSE144130).
( Figure 4B), indicating that the effect of Serpina3c is not at the level of transcription but rather at the level of protein translation or degradation. Nevertheless, knockdown of integrin a5 mRNA inhibited adipocyte differentiation with MDI. With CM treatment, adipogenesis of si-Serpina3c cells was slightly rescued owing to the intracellular Serpina3c protein in CM. In contrast, knockdown of integrin a5, a downstream molecule of Serpina3c, showed no change between MDI and CM treatment ( Figure 4C). Besides transducing signals from the extracellular matrix to the cell, integrin also serves to attach the cell to the extracellular matrix (Gao et al., 2014). Our experiment indicates that knockdown of integrin a5, without altering cellular structure, blocks adipogenesis by inhibiting ERK signaling ( Figure 4D). Confocal microscopy also has shown that the cellular structure of integrin a5 knockdown cells was not changed during differentiation ( Figure S1).
How does Serpina3c contribute to the maintenance of integrin a5 protein? To this end, we measured cathepsin G activity. During adipogenesis, cathepsin G activity was significantly increased during adipocyte differentiation in cells with Serpina3c knockdown ( Figure 4E). Moreover, administration of a synthetic cathepsin G inhibitor during adipogenesis increased PPARg and C/EBPa expression, suggesting that the inhibition of cathepsin G promotes adipogenesis ( Figure 4F). Altogether, these data show that Serpina3c leads to the inhibition of cathepsin G, thereby protecting integrin a5 from degradation. (F) Overexpression vector was generated by inserting mouse Serpina3c (or Serpina3n) encoding a C-terminal FLAG tag into pcDNA3.0. HEK293T cells were transfected with this construct, and 2 days later, the medium was collected, precipitated by cold acetone, and subjected to western blot analysis to detect the FLAG-tagged protein secreted from cells. pcDNA3.0 was used as a negative control. (G) Serpina3c expression and secretion patterns induced by MDI in 3T3 L1 adipocytes. RT-PCR was performed to detect expression of Serpina3c mRNA after treatment. Western blot analysis was performed to investigate expression and secretion of Serpina3c protein in precipitated media or cell lysate after treatment.

Serpina3c Knockdown Attenuates Nuclear Translocation of GSK3b via Prolonged AKT Activation
We next investigated AKT activation, which is also induced by IGF-1 signaling . Knockdown of Serpina3c prolonged the increase in AKT phosphorylation during differentiation ( Figure 5A). Accordingly, phosphorylation of the downstream target glycogen synthase kinase 3b (GSK3b) was also prolonged ( Figure 5A). Phosphorylated GSK3b does not translocate to the nucleus (Tang et al., 2005). Consistent with this, no phosphorylated GSK3b was detected in the nuclear fraction of 3T3-L1 adipocytes, and a decreased amount of nuclear GSK3b was observed in cells with Serpina3c knockdown (Figure 5B), indicating that nuclear translocation was suppressed. The inhibition of GSK3b nuclear translocation by knockdown of Serpina3c was confirmed by immunocytochemistry ( Figure 5C). Thus, Serpina3c promotes GSK3b translocation to the nucleus, which is required for the phosphorylation of Ser184 and Thr179 of C/EBPb that is necessary for adipogenesis (Tang et al., 2005).

Serpina3c Increases Adipogenesis by 3T3-L1 Preadipocytes and Is Increased in Animals and Humans with Obesity
To verify the role of Serpina3c in adipogenesis, the differentiation of adipocytes was examined in cells overexpressing Serpina3c. The expression of C/EBPa and PPARg during differentiation was increased in 3T3-L1 cells transfected with pcDNA3.0 encoding FLAG-tagged Serpina3c ( Figure 6A). Oil Red O staining revealed that the differentiation of these cells was accelerated ( Figure 6B). Consistent with an increase of adipogenesis by Serpina3c, we found that the expression of this factor was higher in epididymal white adipose tissues of mice fed a high-fat diet than in those fed a chow diet ( Figure 6C). When comparing the expression of Serpina3c by the duration of high-fat diet, the Serpi-na3c expression in high-fat diet for 8 weeks was slightly higher than that of 16 weeks but was not statistically significant. We also accessed a public repository (NCBI, Gene Expression Omnibus GDS5056 and GDS1480) to obtain expression data from adipose stem cells from morbidly obese and non-obese individuals. The expression levels of SERPINA3, a human ortholog gene of SERPINA3C, was higher in obese individuals than in lean individuals (p < 0.05; Figure 6D). These data show that SerpinA3 family proteins, including Serpina3c, play a critical role in adipocyte differentiation and obesity. (B) RT-PCR was performed to assess the expression of integrin a5, b3, and a6. 3T3-L1 cells were transfected with NC or Serpina3c siRNA, and then differentiation was induced by MDI. (C) Cells treated with NC, si-Serpina3c (si-3C), or si-Integrin a5 (si-a5) were stained with Oil Red O on day 6, and depletion of Serpina3c or integrin a5 was assessed by RT-PCR. (D) Microscopy images of cells 2 days after transfections with NC and si-a5. Cell structure was not changed. Western blot analysis was performed for ERK and phosphorylated ERK. Scale bar, 50 mm. (E) Cathepsin G activity was measured in 3T3-L1 cells transfected with NC or si-Serpina3c on day 0 or day 1 after differentiation. Data are represented as mean G SD. p Values less than 0.05 were considered significant, with *p < 0.05, **p < 0.01 as determined by Student's t test. (F) Gene expression changes of PPARg and C/EBPa during differentiation after treatment with cathepsin G inhibitor. Data are represented as mean G SD. p Values less than 0.05 were considered significant, with *p < 0.05, **p < 0.01 as determined by Student's t test.

DISCUSSION
The mechanisms of 3T3-L1 differentiation have been investigated since these cells were first cloned by Howard Green (Green and Meuth, 1974). These efforts have identified the roles of transcription factors C/EBPb, C/EBPa, and PPARg in adipogenesis (Gregoire et al., 1998;Lin and Lane, 1994), but the complex molecular processes regulating adipocyte differentiation have yet to be fully defined. In the present study, we found that Serpina3c is a regulator of adipogenesis and was responsible for the accelerated differentiation of adipocytes induced by CM . We further show that its regulation is via modulation of integrin-IGF-1 signaling.
From the RNA sequencing data, the gene expression profile was compared between CM-induced adipocytes and MDI-induced adipocytes, elucidating Serpina3c as a gene expected to be critically involved in adipogenesis. Serpins are the largest and most broadly distributed superfamily of protease inhibitors (Barrett et al., 2001;Irving et al., 2000;Law et al., 2006), nearly ubiquitous in higher eukaryotes and with highly diverse functions. However, the role of serpins in metabolism has not been investigated fully. SerpinB1 was recently found to promote pancreatic b-cell proliferation and contribute to insulin sensitivity (El Ouaamari et al., 2016). With regard to adipocytes, Serpina12 (vaspin [visceral adipose tissue-derived serine protease inhibitor]) is the most well-known serpin, functioning as an insulin-sensitizing adipokine in obesity (Hida et al., 2005). Our data here reveal that Serpina3c stimulates adipocyte differentiation.
Serpina3c is a secretory protein that can affect extracellular matrix proteins. We show here that it is a serine protease inhibitor, with one target protein being cathepsin G for integrin a5 degradation ( Figure 4A). The knockdown of Serpina3c interfered with IGF-1 signaling, disrupting the activation of ERK and AKT. Figure 7 shows a possible mechanism by which Serpina3c controls adipogenesis. IGF-1 receptors form a complex with integrin a5-b3 heterodimers required for activation of the IGF-1 pathway in 3T3-L1 cells. The binding of IGF-1 to its receptor induces the phosphorylation of integrin b3 to bind Src and activate downstream targets, such as ERK. Serine proteases (e.g., cathepsin G) degrade integrin a5 and abolish Src-mediated ERK activation. In the absence of the integrin complex, IGF-1 signals through insulin response substrate 1, which leads to a sustained AKT signaling and the phosphorylation and nuclear export of GSK3b. Thus, (B) Nuclear extracts (NE) and whole-cell lysates were prepared 24 h after MDI treatment and phosphorylated (p) GSK3b and GSK3b levels were measured by western blotting. LaminA/C and GAPDH were used as nuclear and cytosol protein marker, respectively. (C) Cells transfected with NC or si-Serpina3c were induced to differentiate with MDI and fixed for immunocytochemistry against GSKb 24 h later. Scale bar, 50 mm.
Serpina3c is required for activation of ERK and nuclear translocation of GSK3b, which are both are necessary for C/EBPb phosphorylation and adipogenesis.
In summary, the results from this study provide important advances in understanding the mechanism of adipogenesis. We show that CM induces the transcription of several genes, including Serpina3c, which stimulates adipocyte differentiation without mitotic clonal expansion. Furthermore, we show that integrin in the extracellular matrix contributes to adipocyte differentiation. Specifically, Serpina3c inhibits serine proteases that degrade integrin a5, thereby shifting IGF-1 signaling away from AKT activation toward ERK signaling via the IGF-1-integrin complex. Thus, Serpina3c plays an important role in the transition from mitotic clonal expansion to terminal differentiation and is critical for adipocyte differentiation and adipogenesis. (D) SERPINA3 gene expression levels in adipose stem cells or preadipocytes from obese and lean individuals, using publicly available repository data (Gene Expression Omnibus accessions GDS5056 and GDS1480). Data are represented as mean G SD. p Values less than 0.05 were considered significant, with *p < 0.05, **p < 0.01 as determined by Student's t test.

Limitations of the Study
In this study, we suggested cathepsin G as a candidate serine protease that is inhibited by Serpina3c. However, there is a possibility that other proteases under the inhibition of Serpina3c may exist in addition to cathepsin G. Another limitation is that, in humans, Serpina3 clade is consisted of single gene, SERPINA3, so it is necessary to confirm whether SERPINA3 functions in the same mechanism as SERPINA3C in mice. In addition, further study using SERPINA3C knockout mice should be performed to determine how Serpina3c works in vivo. Finally, further purification of Serpina3c protein is needed to fully clarify the effect of Serpina3c overexpression.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.

DATA AND CODE AVAILABILITY
The accession numbers for the 3T3-L1 RNA-seq using MDI and CM is GEO: GSE144130. Expression levels of SERPINA3 in human adipose tissue, which is a human ortholog gene of SERPINA3C, were obtained from a public repository (GEO: GDS5056 and GDS 1480). Expression levels of SERPINA3 were compared among groups by Student's t tests using R version 3.6.0 software (R Development Core Team, Vienna, Austria).

DECLARATION OF INTEREST
The authors declare no conflict of interest. Regulates Adipogenesis (A) IGF-1 receptors form a complex with the integrin a5-b3 heterodimer that is required to activate ERK. With ligand binding, integrin b3 is phosphorylated at Tyr759, leading to Src binding and ERK activation. In this situation, Serpina3c blocks any serine proteases, which can degrade integrin a5. Insulin receptor substrate 1 (IRS-1) may transiently induce AKT signaling. (B) In the absence of Serpina3c, serine proteases degrade integrin a5, and the integrin-IGF-1 receptor complex is not formed, resulting in persistent activation of AKT by IRS-1 and phosphorylation of GSK3b, thereby attenuating adipose differentiation. 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μg/ml of insulin, for 2 days.
Cells were then cultured in DMEM containing 10% FBS and insulin for another 2 days, after which they were grown in DMEM containing 10% FBS. For preparation of CM, 2-day postconfluent 3T3-L1 cells were induced differentiation by MDI for 48 h, after which the used culture medium was harvested .
Oil Red O staining. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% formalin in PBS for 5 min. After rinsing with distilled water, the cells were stained for 1 h in a 60% filtered solution (vol/vol in distilled water) of Oil Red O stain (0.5 g Oil Red O/100 ml isopropanol) and rinsed twice with distilled water.
PCR analysis. Five micrograms of total RNA isolated with Trizol reagent was reverse transcribed with SuperScript II reverse transcriptase using random hexamer primers. The