Prolactin Enhances CCAAT Enhancer-Binding Protein-ß (C/EBPß) and Peroxisome Proliferator-Activated Receptor {gamma} (PPAR{gamma}) Messenger RNA Expression and Stimulates Adipogenic Conversion of NIH-3T3 Cells

Rika Nanbu-Wakao, Yoshio Fujitani, Yasuhiko Masuho, Masa-aki Muramatu and Hiroshi Wakao

Helix Research Institute (R.N.-W., Y.M., M.-a.M., H.W.) Chiba, 292-0812, Japan
The First Department of Internal Medicine (Y.F.) Osaka University Medical School Osaka, 565 Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Extracellular stimuli trigger adipocyte differentiation by inducing the complex cascades of transcription. Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) play crucial roles in this process. Although ectopic expression of these factors in NIH-3T3 cells, a multipotential mesenchymal stem cell line, results in adipogenic conversion, little is known as to hormonal factors that regulate adipogenesis in these cells. In this report we demonstrate that PRL, a lactogenic hormone, enhances C/EBPß and PPAR{gamma} mRNA expression and augments adipogenic conversion of NIH-3T3 cells. Moreover, we show that ectopic expression of the PRL receptor in NIH-3T3 cells results in efficient adipocyte conversion when stimulated with PRL and a PPAR{gamma} ligand, as evidenced by expression of the adipocyte differentiation-specific genes as well as the presence of fat-laden cells. We further demonstrate that signal transducer and activator of transcription 5 (Stat5), a PRL signal transducer, activates aP2 promoter in a PRL-dependent manner. These results suggest that PRL acts as an adipogenesis-enhancing hormone in NIH-3T3 cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Adipogenic differentiation is accompanied by a cessation of mitotic proliferation, morphological changes, and an alteration in gene expression (1, 2). Preadipocyte cell lines such as 3T3-L1 and 3T3-F422A have served as in vitro models to elucidate complex cascades of transcriptional events during the differentiation process. Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and the nuclear hormone receptor peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) are potential regulators of this process, relaying external signals to gene expressions that ultimately lead to adipocyte differentiation.

C/EBP are characterized by a common structure, the presence of a C-terminal leucine zipper for dimerization and basic residues responsible for DNA binding. Among the family of C/EBPs, C/EBP{alpha}, -ß, -{delta}, and CHOP (Gadd153) have been shown to be involved in adipogenesis (3, 4, 5, 6, 7). Chronologically, the expression of C/EBPß and C/EBP{delta} precedes that of C/EBP{alpha} during differentiation of 3T3-L1 cells (8). Evidence that these C/EBPs are crucial regulators of adipogenesis stems in part from the observation that ectopic expression of C/EBPß and, to a lesser extent, C/EBP{delta} results in the conversion of preadipocytes or multipotential mesenchymal stem cells into adipocytes (6). As well, overexpression of a dominant-negative form of C/EBPß inhibits 3T3-L1 cell differentiation (6). C/EBP{alpha} has also been shown, from both antisense and overexpression studies, to play a crucial role in adipogenesis (3, 4, 5). Recent studies using gene-targeted mice support these findings. For example, mice ablated of C/EBP{alpha} suffer significant decreases in both brown adipose tissue (BAT) and in white adipose tissue (WAT) (9). Also, while depletion of either the C/EBPß or {delta} gene results in only a mild perturbation of adipogenic differentiation of primary embryonic fibroblasts and a slight volume loss in epidydimal WAT, the C/EBPß and -{delta} double knockout mice display an almost complete abrogation of adipocyte differentiation as well as severely reduced WAT weight due to a greatly diminished number of adipocytes (10).

PPAR{gamma} is a member of the ligand-stimulated nuclear hormone receptor superfamily (11, 12). PPAR{gamma} plays a central role in adipocyte differentiation. Enforced expression of PPAR{gamma} in multipotential mesenchymal stem cells results in adipogenic conversion in the presence of its ligands/agonists, thiazolidinedione, or prostaglandin (13, 14, 15, 16). In adipocytes, PPAR{gamma} is responsible for the expression of adipose differentiation-related genes such as 422/aP2, phosphoenol pyruvate carboxykinase, and lipoprotein lipase. Indeed, promoters of these genes contain PPAR{gamma} binding sites (17, 18, 19).

Although these families of transcription factors have been shown to play important roles in adipogenesis, little is known about which extracellular stimuli drive the mesenchymal cell to commit toward the adipogenic lineage. In vitro studies have established that hormones such as dexamethasone (DEX), methylisobutylxanthine (MIX), insulin, and those present in FBS trigger the adipogenic conversion of 3T3-L1 cells as well as NIH-3T3 cells ectopically expressing C/EBPs or PPAR{gamma}. MIX and DEX are direct inducers of C/EBPß and {delta}, respectively (8). This induction, in turn, promotes PPAR{gamma} expression and ultimately stimulates adipogenesis (20, 21). In contrast, simple treatment of NIH-3T3 cells with the above mentioned adipogenic hormones does not lead to adipogenic conversion. These observations indicate that some signal transducers are absent or present at low levels in NIH-3T3 cells, and the addition of some hormones or factors that up-regulate the expression of C/EBPs or PPAR{gamma} may have a potential to convert NIH-3T3 cells into adipocytes. Recently, it has been shown that the PRL receptor mRNA is up-regulated during preadipocyte differentiation (22). PRL is best known as a lactogenic hormone responsible for the development of the mammary gland, and it plays a crucial role in reproduction, although many other functions are also reported (23). With regards to diseases, PRL-secreting pituitary adenoma is occasionally associated with obesity (24). In some cases of prolactinoma and obesity, normalization of serum PRL level results in reduction of body weight (25). These observations prompted us to investigate a possible role of PRL and its cognate receptor in adipogenesis. We herein demonstrate that PRL enhances C/EBPß and PPAR{gamma} mRNA production in conjunction with MIX and DEX in NIH-3T3 cells and provide evidence that ectopic expression of the PRL receptor results in adipogenic conversion of NIH-3T3 cells in the presence of a PPAR{gamma} stimulator. We also show that PRL contributes to the activity of aP2 gene promoter via signal transducer and activator of transcription 5 (Stat5).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL Augments C/EBPß Transcription in Multipotential Mesenchymal Stem Cells (NIH-3T3 Cells)
Since C/EBPß and {delta} are the initial transcription factors required for adipogenesis and their mRNAs are induced by a variety of stimuli, including MIX, DEX, insulin, lipopolysaccharide, interleukin (IL)-1, IL-6, and GH (8, 26, 27), we first asked whether PRL enhances C/EBPß mRNA expression in multipotential mesenchymal stem cells (NIH-3T3) and in 3T3-L1 preadipocyte cells. Confluent NIH-3T3 or 3T3-L1 cells were treated with or without increasing concentrations of PRL or with various effectors, and Northern blot analysis was performed (Fig. 1Go). While nontreated NIH-3T3 cells expressed a low level of C/EBPß mRNA, PRL increased its transcript in a dose-dependent manner (Fig. 1AGo, lanes 1–5). At 333 ng/ml of PRL, the induction was saturated (Fig. 1AGo, lane 4). This dose of PRL was as efficacious as MIX, DEX, and insulin (Fig. 1AGo, lanes 4 and 6–8). FBS was not as potent as PRL (Fig. 1AGo, lane 9). The effect of PRL was also examined in 3T3-L1 cells. PRL failed to enhance C/EBPß mRNA (Fig. 1BGo, lanes 1–5), while MIX, DEX, and insulin increased C/EBPß mRNA expression as previously reported (Fig. 1BGo, lanes 6–8) (8). FBS moderately enhanced its expression but less efficiently than the other stimulators (Fig. 1BGo, lane 9). No PRL-dependent expression of C/EBP{delta} mRNA was detected in either cell type (data not shown). These results suggest that PRL has an inductive effect on C/EBPß transcription, at least in NIH-3T3 cells.



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Figure 1. Induction of C/EBPß mRNA by Different Effectors in NIH-3T3 Cells (A) and in 3T3-L1 Preadipocytes (B)

Confluent cells were exposed to different effectors in DMEM containing 10% CS (lanes 1–8) or to DMEM containing 10% FBS (lane 9) for 3 h. No effector (lane 1), different concentrations of PRL (P; lane 2, 37 ng/ml; lane 3, 111 ng/ml; lane 4, 333 ng/ml; lane 5, 1 µg/ml), MIX (lane 6, M; 0.5 mM), DEX (lane 7, D; 1 µM), and insulin (lane 8, I; 10 µg/ml). Total RNA was analyzed by Northern blot hybridization using the DIG-labeled antisense C/EBPß RNA. An equivalent amount of total RNA (8 µg/lane) was loaded as indicated by staining of ribosomal RNA.

 
PRL Enhances PPAR{gamma} mRNA in NIH-3T3 Cells as Well as in Preadipocyte 3T3-L1 Cells
Since ectopic expression of C/EBPß in multipotential mesenchymal stem cells results in activation of PPAR{gamma} expression (20), we next asked whether PRL might influence PPAR{gamma} expression. As optimal PPAR{gamma} mRNA production requires MIX, DEX, and insulin (21), PRL was challenged together with these adipogenic hormones. Figure 2Go shows Northern blot analysis for NIH-3T3 (Fig. 2AGo) and 3T3-L1 cells (Fig. 2BGo). While PPAR{gamma} mRNA was efficiently induced by MIX, DEX, and insulin in 3T3-L1 cells, it was barely detected in NIH-3T3 cells in the absence of PRL (Fig. 2Go, A and B, lane 1). In both cell lines, however, addition of PRL resulted in a dose-dependent transcriptional augmentation (Fig. 2Go, A and B, lanes 2–5). PRL at the concentration of 333 ng/ml was as potent as FBS in both cell lines (Fig. 2Go, A and B, lanes 4 and 6). To further determine which adipogenic effector(s) synergize with PRL for PPAR{gamma} mRNA expression, different combinations were tested in NIH-3T3 (Fig. 2CGo) and in 3T3-L1 cells (Fig. 2DGo). MIX, DEX, and insulin efficiently induced PPAR{gamma} mRNA in 3T3-L1 cells but much less efficiently in NIH-3T3 cells (Fig. 2Go, D and C, lane 1). In NIH-3T3 cells PRL alone or in combination with MIX, insulin, or DEX induced little mRNA (Fig. 2CGo, lanes 2–5). Combination of MIX and DEX, but not MIX and insulin, nor DEX and insulin, led to strong PPAR{gamma} mRNA expression together with PRL (Fig. 2CGo, lanes 6–8). Insulin plus MIX and DEX resulted in a maximized induction, and FBS was as efficacious as PRL (Fig. 2CGo, lanes 9 and 10). Similar results were obtained in 3T3-L1 cells (Fig. 2DGo). It is noteworthy that DEX and PRL had an inductive effect, which was slightly enhanced by insulin (Fig. 2DGo, lanes 5 and 8). These data demonstrate that PRL enhances MIX- and DEX-induced PPAR{gamma} expression in both cell lines.



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Figure 2. PRL Dose-Dependent Enhancement of PPAR{gamma} mRNA Expression in NIH-3T3 Cells (A) and in 3T3-L1 Preadipocytes (B)

Confluent cells were exposed to DMEM containing 10% CS, insulin, DEX, and MIX for 48 h with increasing concentrations of PRL (lane 1, none; lane 2, 37 ng/ml; lane 3, 111 ng/ml; lane 4, 333 ng/ml; lane 5, 1 µg/ml). As a control, confluent cells were incubated with 10% FBS in the presence of the same cocktail (lane 6). Total RNA (4 µg/lane) was analyzed by Northern blot hybridization with the DIG-labeled antisense PPAR{gamma} RNA. Panels C and D, Induction of PPAR{gamma} mRNA by different adipogenic effectors in NIH-3T3 cells (C) and in 3T3-L1 preadipocytes (D). Confluent cells were exposed to various combinations of effectors, MIX (M; 0.5 mM), DEX (D; 1 µM), insulin (I; 10 µg/ml), and PRL (P; 1 µg/ml) in DMEM containing 10% CS (lanes 1–9) for 48 h. As a control, 10% FBS plus MIX, DEX, and insulin were included (lane 10). Total RNA was extracted, and 3 µg of each sample were subjected to Northern blot analysis.

 
Ectopic Expression of the PRL Receptor Results in Enhanced Adipogenic Conversion of NIH-3T3 Cells
The above data suggested that PRL might endow NIH-3T3 cells with the ability to convert toward adipocytes. However, no terminal differentiation marker gene expression or morphological changes were observed even after 12 days of incubation with PRL under the normal permissive conditions (data not shown). Since NIH-3T3 cells do not undergo adipogenic conversion as readily as 3T3-L1 preadipocyte cells in normal permissive medium, we employed strong permissive conditions using troglitazone, a ligand of PPAR{gamma}. Troglitazone is a member of thiazolidinediones, which has been shown to elevate the potential of multipotential mesenchymal cells to differentiate (14, 28). Under these conditions, we set out to study the function of PRL in adipogenic conversion of NIH-3T3 cells. The PRL receptor gene was stably transfected into NIH-3T3 cells along with the neomycin resistance gene. As a control, cells harboring only the neomycin resistance gene were also selected. G418-resistant colonies were cultured for 2 weeks and were induced to differentiate in situ. Before differentiation stimulation, no fat-laden differentiated cells were observed (data not shown). However, upon exposure to the strong permissive regimen for 10 days, some terminally differentiated adipocyte colonies, as evidenced by Oil-Red-O staining, appeared in the plates transfected with the PRL receptor (Fig. 3BGo, PRLR). From three independent experiments, 11% of the G418-resistant colonies cotransfected with the PRL receptor gave rise to differentiated adipocytes. In contrast, 2% of differentiated colonies were present in the control cells (Fig. 3AGo, Neo). The omission of PRL from the medium resulted in a significant decrease of differentiated colony number in both cases (data not shown). These data further reinforced our hypothesis that PRL and the PRL receptor play an important role in adipogenic differentiation of NIH-3T3 cells.



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Figure 3. Effect of the PRL Receptor Expression on Colonies of NIH-3T3 Cells Exposed to the Adipogenic Hormones

NIH-3T3 cells were transfected with a neomycin resistance gene expression vector (pSV2neo) alone (A, Neo) or together with a PRL receptor expression vector (B, PRLR). G-418-resistant colonies (60–70 per dish) were tested for differentiation in situ. Colonies with tightly packed cells were exposed to the strong permissive regimen in the presence of 1 µg/ml of PRL for 10 days, and subsequently fixed and stained with Oil-Red-O. Red colonies correspond to clones that showed substantive evidence of fat accumulation when monitored by light microscopy.

 
To further confirm these observations, stable NIH-3T3 cells ectopically expressing the PRL receptor or the neomycin resistance gene alone were selected by G418, and more than 20,000 individual clones were pooled. Expression of the exogenous PRL receptor mRNA in transfectant cells was confirmed by Northern blot hybridization (data not shown). Pooled cells were used to avoid possible clonal variation in the ability to commit differentiation (29). First, we examined the effect of the PRL receptor on C/EBPß and PPAR{gamma} mRNA expression. Cells transfected only with the neomycin resistance gene exhibited similar dose-dependent C/EBPß mRNA expression as parental cells (Figs. 1AGo and Fig. 4AGo, lanes 1–5). In contrast, the PRL receptor-expressing cells showed an enhanced sensitivity to a given dose of PRL (Fig. 5AGo, lanes 6–10). Even at the highest concentration of PRL (1 µg/ml), the C/EBPß mRNA level in cells expressing the neomycin resistance gene was lower than that induced with 111 ng/ml of PRL in the PRL receptor-expressing cells (Fig. 4AGo, compare lanes 5 and 8). As for PPAR{gamma} mRNA, the same PRL dose dependency was observed in parental and in the control cells (Figs. 2AGo and Fig. 4BGo, lanes 1–5). Interestingly, ectopic expression of the PRL receptor made these cells much more sensitive to PRL as in the case for C/EBPß mRNA (Fig. 4BGo, lanes 1–5 and 6–10). Addition of 111 ng/ml of PRL to the PRL receptor-expressing cells resulted in the similar induction of PPAR{gamma} mRNA as seen with 1 µg/ml of PRL to the control cells (Fig. 4BGo, compare lanes 5 and 8). We next examined the differentiation program of these two cells with or without PRL (Fig. 5Go). PRL slightly elevated C/EBPß mRNA in the control cells after 3 days (Fig. 5AGo). In the PRL receptor-overexpressing cells, further up-regulation was detected especially at day 3 (compare lanes 4 and 9 in Fig. 5AGo and those in Fig. 5BGo), and thereafter PRL sustained C/EBPß mRNA at a slightly higher level (Fig. 5Go, A and B, compare lanes 9–11). As for C/EBP{delta} mRNA, addition of PRL had no effect in either cell throughout the entire differentiation process (data not shown). Expression of PPAR{gamma} mRNA peaked on day 2 in the control cells, and then sharply declined in the absence of PRL (Fig. 5AGo, lanes 1–6). Ectopic expression of the PRL receptor resulted in enhanced expression, even in the absence of exogenous PRL (Fig. 5BGo, lane 3). Addition of PRL kept the level of PPAR{gamma} mRNA slightly high during the later period of the differentiation program in both control and PRL receptor-overexpressing cells (Fig. 5Go, A and B, lanes 9–11). Adipsin mRNA was detected on day 5 in both control and PRL receptor-overexpressing cells, and PRL significantly enhanced the mRNA in the PRL receptor-overexpressing cells (Fig. 5Go, A and B, lanes 5 and 10). Quite interestingly, the control cells failed to accumulate adipsin mRNA by day 8, while the PRL receptor-overexpressing cells kept adipsin mRNA high until day 8 (Fig. 5Go, A and B, lanes 5, 6, 10, and 11). aP2 mRNA was detected as early as day 3 in both control and PRL receptor-overexpressing cells (lanes 4 in both panels A and B). Addition of PRL strongly augmented the expression of aP2 mRNA, particularly in cells overexpressing the PRL receptor (lanes 9–11 in both panels A and B). Glycerol-3-phosphate dehydrogenase (GPD) mRNA was detected on day 8 in the control cells; however, the effect of PRL on the mRNA was not clear (panel A, lanes 6 and 11). On the other hand, PRL increased GPD mRNA in cells overexpressing the PRL receptor (Fig. 5BGo, lanes 5, 6, 10, and 11). We have repeated these experiments four times and obtained essentially identical results.



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Figure 4. Effect of Ectopic Expression of the PRL Receptor on C/EBPß and PPAR{gamma} mRNA in NIH-3T3 Cells

A, Enhanced induction of C/EBPß mRNA by ectopic expression of the PRL receptor. NIH-3T3 cells were transfected with pSV2neo alone (Neo) or PRL receptor expression vector together with pSV2neo (PRLR). Resulting G-418-resistant stable clones were pooled and grown to confluence, and then exposed to increasing concentrations of PRL (P) (lanes 1 and 6, no ligand; lanes 2 and 7, 37 ng/ml; lanes 3 and 8, 111 ng/ml; lanes 4 and 9, 333 ng/ml; lanes 5 and 10, 1 µg/ml) in DMEM containing 10% CS for 3 h. Total RNA was extracted, and 3 µg of each sample were subjected to Northern blot analysis. B, Enhancement of PPAR{gamma} mRNA by ectopic expression of the PRL receptor. Confluent cells prepared as described above were exposed to different concentrations of PRL (P) in DMEM containing 10% CS, insulin, DEX, and MIX for 48 h. The concentrations of PRL were the same as in panel A. Total RNA (2 µg/lane) was analyzed by Northern blot hybridization.

 


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Figure 5. Effect of PRL and the PRL Receptor on Adipogenic Gene Expression in NIH-3T3 Cells

A, Expression of adipogenic genes in cells expressing only the neomycin resistance gene (Neo). B, Expression of adipogenic genes in cells ectopically expressing the PRL receptor gene (PRLR). G-418-resistant stable clones were pooled and grown to confluence, and then exposed to DMEM containing 10% FBS together with insulin, DEX, and MIX for 48 h in the absence (FBS+I+T) or presence of 1 µg/ml of PRL (FBS+I+T+PRL). Cells were then maintained in DMEM containing 10% FBS, 2.5 µg/ml of insulin, and 5 µM of troglitazone in the absence (FBS+I+T) or presence of 1 µg/ml of PRL (FBS+I+T+PRL) and replenished with this medium every other day. Total RNA was isolated at the indicated time point, and 3 µg of each sample were analyzed by Northern blot hybridization using the indicated DIG-labeled antisense riboprobes.

 
Both cells were then stained with Oil-Red-O after 10 days of the adipocyte-inducing regimen. While virtually no differentiation was observed in the absence of PRL (Fig. 6AGo, Neo, FBS+I+T), 4% of control cells exhibited adipogenic conversion in the presence of PRL as judged by Oil-Red-O-positive cell number (Fig. 6AGo, Neo, FBS+I+T+PRL). In contrast, 18% of the PRL receptor-transfected cells underwent adipogenic conversion in the presence of PRL, as assessed by the formation of fat droplets in cells (Fig. 6BGo, PRLR, FBS+I+T+PRL). On the other hand, omission of PRL resulted in a significant decrease of differentiated cell population to 5% (Fig. 6BGo, PRLR, FBS+I+T). Four independent experiments were performed and 13–23% of G-418-resistant cells overexpressing the PRL receptor converted into lipid-laden adipocytes in the presence of PRL. In light of these results, we concluded that PRL and its cognate receptor up-regulate the adipogenic conversion of NIH-3T3 multipotential mesenchymal stem cells.



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Figure 6. Effect of PRL and the PRL Receptor on Adipogenic Differentiation of NIH-3T3 Cells

A pool of NIH-3T3 cells expressing only the neomycin resistance gene (A, Neo) or the PRL receptor gene (B, PRLR) were grown to confluence and exposed to the strong permissive conditions as described in Fig. 5Go in the absence (FBS+I+T) or presence of 1 µg/ml of PRL (FBS+I+T+PRL) for 10 days. Cells were fixed and stained with Oil-Red-O. Original magnification, x10.

 
Stat5 Activates aP2 Promoter
Finally, we examined a possible molecular mechanism by which PRL enhances the adipogenic conversion, particularly the induction of the adipocyte-specific genes. To assess the role of PRL in adipogenesis, we used the aP2 promoter as a model system. Since PRL activates Stat5 (30), we examined the effects of Stat5 on the aP2 promoter. To this end, the aP2 promoter construct was transfected into NIH-3T3 cells without or with murine Stat5A, PPAR{gamma}2, Stat5A plus PPAR{gamma}2, or an ovine dominant negative form of Stat5(Y694F) (31). After transfection cells were treated with different combinations of PRL and troglitazone (a PPAR{gamma} ligand). When the aP2 promoter alone was transfected, any combination of PRL/troglitazone failed to enhance the promoter activity (Fig. 7Go, lanes 1–4). In contrast, transfection with Stat5A cDNA resulted in a PRL-dependent 2-fold increase in promoter activity (Fig. 7Go, lanes 7 and 8). Troglitazone did not affect the promoter activity (Fig. 7Go, lane 6). When PPAR{gamma}2 cDNA was cotransfected, troglitazone-dependent enhancement of the promoter activity was observed. Challenge with troglitazone resulted in 60% augmentation, while combination with PRL gave 100% increase in promoter activity (Fig. 7Go, lanes 9, 10, and 12). PRL alone had no effect in this case (Fig. 7Go, lane 11). Cotransfection with both Stat5A and PPAR{gamma}2 cDNA showed additive effects. Troglitazone or PRL challenge doubled the promoter activity, whereas the combination of both ligands resulted in a 2.5-fold increase (Fig. 7Go, lanes 13–16). These data strongly suggested that Stat5 regulates aP2 promoter activity. To further strengthen this hypothesis, ovine dominant negative Stat5 (Y694) was transfected into cells together with aP2 promoter. When challenged with PRL, no ligand-dependent promoter activity was observed (Fig. 7Go, lanes 17, 18, and 19). Addition of troglitazone had no effect on promoter activity (Fig. 7Go, lanes 18 and 20). Use of Stat5B and the corresponding dominant negative Stat5B (Y699F) gave essentially identical results (data not shown). These data indicate that the aP2 gene is regulated by PRL, at least partially, via Stat5.



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Figure 7. PRL Contributes to the aP2 Promoter Activity via Stat5

A luciferase reporter gene linked to the aP2 promoter pGL2-aP2 was transfected into parental NIH-3T3 cells along with or without the expression vectors (cDNA). After treatment with the indicated ligand(s) for 6 h, cells were harvested and both firefly and renilla luciferase activities were measured. Relative luciferase activity is shown. SD is indicated by the error bar. Lanes 1–4, No expression vector; lanes 5–8, murine Stat5A expression vector; lanes 9–12, murine PPAR{gamma}2 expression vector; lanes 13–16, murine Stat5A and PPAR{gamma}2 expression vectors; lanes 17–20, dominant negative ovine Stat5(Y694F) expression vector. Lanes 1, 5, 9, 13, and 17, no ligand; lanes, 2, 6, 10, 14, and 18, troglitazone (5 µM); lanes 3, 7, 11, 15, and 19, PRL (1 µg/ml); lanes 4, 8, 12, 16, and 20, troglitazone+PRL.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report we provide evidence that 1) PRL augments the expression of C/EBPß and PPAR{gamma} mRNA in conjunction with MIX and DEX in NIH-3T3 cells. 2) PRL enhances the adipogenic conversion of NIH-3T3 cells under strong permissive conditions (i.e. in the presence of a PPAR{gamma} ligand/agonist). 3) ectopic expression of the PRL receptor efficiently converts NIH-3T3 cells into fat-laden adipocytes. 4) PRL regulates aP2 promoter via Stat5.

3T3-L1 cells are of preadipocyte lineage and differentiate into adipocytes when stimulated appropriately (1). Growth factors such as insulin, insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor have been shown to promote adipogenesis in 3T3-L1 cells, and, in fact, FBS is a potent stimulus (32, 33, 34). On the other hand, NIH-3T3 cells are considered to be a multipotential mesenchymal cell line that does not readily differentiate into adipocytes as 3T3-L1 cells do. Nevertheless, NIH-3T3 cells do convert into adipocytes when transcription factors such as C/EBPs, PPAR{gamma}, and ADD-1 are overexpressed (4, 6, 13, 35). These data imply that NIH-3T3 cells possess a potential to undergo adipogenesis. To our knowledge, there has been no report of any growth factor or cytokine that enhances adipogenesis in NIH-3T3 cells. Our present data clearly show that PRL and its cognate receptor are required but not sufficient for the activation of adipogenic program in NIH-3T3 cells. While PRL increased C/EBPß mRNA in a concentration-dependent fashion in NIH-3T3 cells, no such effect was observed in 3T3-L1 preadipocytes. At present, the reason for this difference is unclear. In both NIH-3T3 and 3T3-L1 cells, PRL enhanced PPAR{gamma} mRNA expression in conjunction with other adipogenic hormones such as MIX and DEX (Fig. 2Go, A–D). As MIX and DEX induce C/EBPß and -{delta}, respectively (8), it is conceivable that there is a cooperation between PRL-emanated signals and those of C/EBPß and/or -{delta} for the maximum production of PPAR{gamma} mRNA. Further study is required to fully understand how PRL receptor-triggered signals act together with these transcription factors. The fact that FBS was as potent as PRL in inducing C/EBPß and PPAR{gamma} mRNA suggests that FBS contains PRL, or that some factors present in FBS enhance PPAR{gamma} mRNA (Figs. 1AGo and 2CGo and 2D). The report that FBS is rich in PRL rather supports the former possibility (36). The presence of PRL in FBS may explain the relatively high level of PPAR{gamma} mRNA expression in the PRL receptor-overexpressing cells in the absence of exogenous PRL (Fig. 5BGo, lane 3). This, in turn, may contribute the slight increase in the number of differentiated cells in cells overexpressing PRL receptors (Fig. 6BGo, PRLR, FBS+I+T).

PRL has been shown to induce calcium influx and trigger activation of signaling molecules such as Ras, PI3 kinase, and the STATs (23, 30, 37, 38). It remains to be determined which of these signaling pathways are responsible for C/EBPß and PPAR{gamma} expression. As for the transcriptional regulation of PPAR{gamma} mRNA, it has been shown that granulocyte-colony stimulating factor (G-CSF), 12-O-tetradecanoylphorbol-13-acetate, 1,25-dihydroxyvitamin D3, and oxidized low density lipoprotein promote its transcription in macrophages (39, 40). Since both G-CSF and PRL activate JAK-STAT pathway, it is tempting to speculate that the maximum induction of PPAR{gamma} mRNA may be dependent on this pathway.

PRL has a potential in enhancing PPAR{gamma} transcript in parental NIH-3T3 cells as well as the PRL receptor-transfected cells (Fig. 5Go). This enhancement may, in part, explain why NIH-3T3 cells receiving PRL and troglitazone exhibit morphological changes and express adipocyte-specific marker genes such as adipsin, aP2, and GPD. Intriguingly, PPAR{gamma} mRNA level peaked at day 2 and gradually declined during the adipogenic conversion (Fig. 5Go). This is in contrast to the observation during the differentiation process of 3T3-L1 cells or NIH-3T3 cells ectopically expressing C/EBPß (17, 20). In these cells a high level of PPAR{gamma} mRNA is sustained at later steps in differentiation. Our data suggest that transient PPAR{gamma} mRNA production is sufficient for the induction of some terminal marker genes, i.e. adipsin, aP2, and GPD and for the accumulation of fat droplets (Figs. 5BGo and 6Go). Why do cells treated with PRL promote adipogenic conversion to a certain extent even though the increase of PPAR{gamma} mRNA is rather transient? A possible explanation is that PRL-elicited signals and those emanated from the activated PPAR{gamma} might coordinate to stimulate adipogenic program. In agreement with this hypothesis, a significant drop of adipsin, aP2, and GPD mRNAs as well as of Oil-Red-O positive cell number was observed in the absence of PRL, while the PPAR{gamma} mRNA expression profile remained nearly identical in the PRL receptor-expressing cells (Figs. 5BGo and 6Go). Our data from aP2 promoter analysis further indicate that aP2 gene is controlled, at least in part, by Stat5, which is activated by PRL (Fig. 7Go). These results also suggest that the effect of PPAR{gamma} and Stat5 on the aP2 promoter is ligand dependent and additive. However, it has yet to be elucidated how Stat5 regulates the aP2 promoter activity. Recently, a cross-talk between PPAR{alpha}, another member of PPAR family, and STAT5B in GH signaling has been shown in COS cells (41). It will be interesting to examine whether there are other adipocyte-specific genes regulated by both PPAR{gamma} and STAT5 as shown here for the aP2 gene.

It is noteworthy that GH shows opposite effects on the expression of adipocyte-specific genes such as PPAR{gamma}, aP2, and fatty acid synthase and on adipogenesis in primary preadipocytes (42). Whether PRL exhibits stimulatory or inhibitory effects on primary preadipocytes remains to be addressed. GH deficiency is often associated with obesity (43), while some prolactinoma patients are obese (24). In both cases, treatment with either GH or with reagents lowing serum PRL results in improvement of obesity (25, 43). These data imply that the action of GH and PRL is opposite in vivo. Given the fact that GH and PRL share many signaling molecules such as Jak2 and Stat5A and B, it is necessary to delineate molecular mechanisms underling these opposing effects.

PRL gene knockout mice grow normally but fail to develop mammary glands (44). PRL receptor gene ablation leads to a similar but somewhat different phenotype, resulting in reproductive defects (45). In both cases, gene-targeted mice grow as wild type. While mice lacking STAT5A grow normally, STAT5B-deficient male mice manifest a significant decrease in weight (46, 47). The STAT5A/STAT5B double-knockout mice exhibit more severe growth impairment, and the size of the fat pads is decreased to one-fifth of the wild type (48). These data suggest that STAT5A and B, both of which are activated by PRL, play a key role in the development of the fat pad. The results presented herein appear to be in line with these observations. The reason why mice lacking PRL or the PRL receptor do not manifest weight loss, as observed in STAT5B or STAT5A/STAT5B knockout mice, can be explained as follows. In the absence of PRL or its cognate receptors, other factor(s) or receptor(s) that activate STAT5A and/or B compensate for their functions. The redundancy in cytokine signaling may mask all of the PRL/PRL receptor functions from gene targeting experiments.

In summary, we have shown here that PRL enhances adipocyte differentiation of NIH-3T3 fibroblasts. Our results may have important implications for defining hormonal stimulation along the adipocyte differentiation pathway. Understanding the interactions between PRL-triggered signals and those emanated from other adipogenic inducers should provide valuable insight into the mechanisms that control adipogenic differentiation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ovine PRL, bovine insulin, and DEX were purchased from Sigma (St. Louis, MO), and methylisobutylxanthine was purchased from Wako Chemical Co (Osaka, Japan). Troglitazone was provided from Sankyo Co., Ltd. (Tokyo, Japan).

Plasmids
The cDNAs for C/EBP{alpha}, C/EBPß, C/EBP{delta}, PPAR{gamma}, aP2, and GPD used in this study were isolated from a 3T3-L1 preadipocyte library constructed 9 days after induction of differentiation, using SuperScript {lambda} system (Life Technologies, Inc., Gaithersburg, MD). The sequences of the cDNAs were verified by DNA sequencing (ABI 377 DNA sequencer). aP2 promoter comprising a -1 to -5.4 kbp fragment was inserted into the SmaI site of pGL2-basic vector (Promega Corp., Madison, WI).

Cell Culture and Induction of Differentiation
3T3-L1 preadipocytes (NIHS cell bank, Tokyo, Japan, catalogue number JCRB9014) and multipotential mesenchymal stem cells (NIH-3T3; Riken cell bank, Tsukuba, Japan; catalogue no. RCB0150) were maintained in growth medium consisting of DMEM (Nisseiken, Kyoto, Japan) containing 10% normal calf serum (CS; Life Technologies, Inc.). To establish stable cell lines, NIH-3T3 cells were transfected with the rat PRL receptor (PRLR) in pME18S and pSV2neo with lipofectAMINE-PLUS reagent following the protocol provided by the supplier (Life Technologies, Inc.). Briefly, a mixture of 8 µg of PRLR plasmids, 0.4 µg of pSV2neo plasmids, 20 µl of PLUS reagent, and 30 µl of lipofectAMINE in 1.5 ml of OPTI-MEM I (Life Technologies, Inc.) was incubated at room temperature for 30 min and then diluted to 8 ml with OPTI-MEM I. Cells plated at a density of 8 x105 cells per 10-cm dish the day before transfection were washed with OPTI-MEM I, and the diluted DNA-lipid mixture was added. After 3 h of incubation at 37 C, cells were refed with DMEM supplemented with 10% normal calf serum and were cultured for an additional 24 h before selection with 0.4 mg/ml of G418. The transfectants were selected for 14 days and subjected to in situ colony differentiation assay. In some experiments, more than 20,000 G418-resistant clones were pooled and assayed for their ability to differentiate. To convert NIH-3T3 cells and the transfectants into adipocytes, cells were grown to confluence (considered as day 0). At this stage, cells were exposed to fresh DMEM containing 10% FBS (Life Technologies, Inc.), 1 µM DEX, 0.5 mM MIX, and 10 µg/ml of insulin for 48 h. After this treatment, the medium was replaced with DMEM containing 10% FBS, and 2.5 µg/ml of insulin, and cells were refed every other day (normal permissive conditions). For strong permissive conditions, 5 µM of troglitazone was included. To distinguish the effect of PRL, 10% CS instead of 10% FBS was used in some experiments, as indicated in the text and figure legends. Adipocyte conversion was assessed by the presence of accumulated fat droplets in the cytoplasts. Cells were fixed with 2% formaldehyde, 0.2% glutaraldehyde in PBS and stained with Oil-Red-O (49).

Northern Blot Analysis
Total RNA was isolated according to the method of Chomczynski and Sacchi (50). The indicated amount of the total RNA was fractionated using 1% agarose/2.2 M formaldehyde gel electrophoresis and was transferred to a nylon membrane (51). rRNA was stained on the filters with methylene blue to assess RNA loading and transfer efficiency (52). The DIG-labeled RNA probes were transcribed from EcoRI-linearized cDNA plasmid in pZL1 according to the Roche Molecular Biochemicals protocol. Hybridization was performed with DIG-labeled antisense RNA probes according to the protocol provided by the supplier (Roche Molecular Biochemicals, Indianapolis, IN).

Transcriptional Activation Assay
The expression vectors for murine Stat5A and ovine Stat5(Y694F) were constructed by ligating the EcoRI-NotI fragment containing Stat5A or Stat5(Y694F) into the EcoRI-NotI site of pME18S. The SalI-NotI fragment comprising PPAR{gamma}2 was inserted into the XhoI-NotI site of pME18S. aP2 promoter-luciferase vector (pGL2-aP2) was transiently transfected into NIH-3T3 cells without or with expression vectors. NIH-3T3 cells were maintained in DMEM containing 10% CS and transfected at 70% confluency with LipofectAMINE-PLUS reagent (Life Technologies, Inc.). A renilla luciferase expression vector (pRL-CMV) was cotransfected as a control for the transfection efficiency (Promega Corp.). After DNA removal by washing, cells were serum starved for 16 h in OPTI-MEM I, and then left untreated or challenged with PRL, troglitazone, or PRL plus troglitazone for 6 h. After cell lysis, both firefly and renilla luciferase activities were measured. Transfections were performed in duplicate and repeated two to four times.


    ACKNOWLEDGMENTS
 
We thank Ms. C. Oda and Y. Kojima for technical assistance and Prof. G. Krystal (The Terry Fox Laboratory, Vancouver, Canada) for critical reading of this manuscript.

Helix Research Institute is supported by the Ministry of International Trade and Industry (MITI), Chugai Pharmaceutical Co., Fujisawa Pharmaceutical Co., Hitachi Co., Mitsubishi Chemical Co., Nippon Godou Finance Co., Kyowa Hakko Co., Sumitomo Chemical Co., Taisho Pharmaceutical Co., Yamanouchi Pharmaceutical Co., and Yoshitomi Pharmaceutical Co.


    FOOTNOTES
 
Address requests for reprints to: Hiroshi Wakao, Helix Research Institute, 1532–3 Yana Kisarazu-shi Chiba, 292-0812, Japan.

Received for publication July 21, 1999. Revision received September 30, 1999. Accepted for publication November 11, 1999.


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