Glucocorticoid Receptor β Stimulates Akt1 Growth Pathway by Attenuation of PTEN*

Background: The glucocorticoid receptor β (GRβ) is a positive regulator of growth. Results: GRβ suppression of PTEN resulted in enhanced phosphorylation of Akt and growth. Conclusion: GRβ enhances insulin-induced proliferation by suppressing PTEN and activating Akt1. Significance: GRβ suppression of PTEN indicates that it has an important role in growth factor signaling and potentially cancer. Glucocorticoids (GCs) are known inhibitors of proliferation and are commonly prescribed to cancer patients to inhibit tumor growth and induce apoptosis via the glucocorticoid receptor (GR). Because of alternative splicing, the GR exists as two isoforms, GRα and GRβ. The growth inhibitory actions of GCs are mediated via GRα, a hormone-induced transcription factor. The GRβ isoform, however, lacks helix 12 of the ligand-binding domain and cannot bind GCs. While we have previously shown that GRβ mRNA is responsive to insulin, the role of GRβ in insulin signaling and growth pathways is unknown. In the present study, we show that GRβ suppresses PTEN expression, leading to enhanced insulin-stimulated growth. These characteristics were independent of the inhibitory qualities that have been reported for GRβ on GRα. Additionally, we found that GRβ increased phosphorylation of Akt basally, which was further amplified following insulin treatment. In particular, GRβ specifically targets Akt1 in growth pathways. Our results demonstrate that the GRβ/Akt1 axis is a major player in insulin-stimulated growth.

Regulation of growth pathways is an important component of cellular physiology that maintains division, nutrient uptake, and cell fate. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) 2 is essential for growth regulation, and its inactivation or deletion has been shown in a variety of cancers (1). A prominent feature of PTEN signaling is inhibition of the phosphoinositide 3-kinase (PI3-kinase) pathway, which results in reduced 3-phosphoinositide (PIP 3 ) levels and suppressed growth (2). A plethora of growth factors and cytokines activate the PI3-kinase lipid enzyme, resulting in the conversion of 2-phosphoinositide (PIP 2 ) to 3-phosphoinositide (PIP 3 ) (3). PIP 3 binds to the PH domain of Akt resulting in phosphorylation and activation, which increases cell survival, growth, and metabolism (2,3). Interestingly, glucocorticoids (GCs), which inhibit growth (4) and induce apoptosis in a variety of cells (5), have been shown to increase expression of PTEN in A549 human lung carcinoma cells (6). This suggests that one aspect of the anti-growth properties of GCs may be mediated through the up-regulation of PTEN. However, the roles of the glucocorticoid receptors (GRs), especially the separation of GR␣ and GR␤ isoforms, in the regulation of PTEN-mediated effects on growth pathways, have not been investigated.
As a result of alternative splicing of exon 9 of a single gene, there exists two GR isoforms, ␣ and ␤, which vary at their C terminus. There also exist a GR␥ isoform from the use of an alternative splice donor site in which three base pairs are retained between exons 3 and 4 (7). As a consequence, an additional arginine is located in the DNA binding domain, resulting in decreased binding (8). The GR ␣ and ␤ isoforms have been the most studied. The GR␣ is a hormone-activated transcription factor that controls many physiological processes, extending from apoptosis, glucose metabolism to lung development (9,10). The GR␤ isoform, however, lacks helix 12 of the C terminus in the ligand-binding domain and cannot bind GCs (11). The anti-proliferative properties of GCs are mediated by GR␣, which have been attributed to elevation of cell cycle arrest proteins, p27 and p21 (12,13). A reduction in GR␣ in murine macrophages results in enhanced growth and decreased expression of p27 (14). This implies that resistance to GC-induced apoptosis may occur by reducing GR␣ levels. In humans, GC resistance can occur by two major mechanisms: loss-of-function mutations in GR␣ (15), or by increased expression of GR␤, which acts as a dominant-negative inhibitor to GR␣ (11). Although GR␣ mutations can result in a type of GC resistance that is both systemic and severe, these mutations are rare. In contrast, the evolving evidence suggests that GC resistance based on GR␤ is much more common and likely to be tissue-specific in nature.
The best-studied apoptotic roles of GCs have been in cancers of the immune system such as lymphoma and leukemia. Immune system homeostasis is balanced by GCs, which regulate immune cell turnover by suppressing cytokine production and promoting apoptosis. GC insensitivity due to elevated human GR␤ (hGR␤) expression results in higher levels of proinflammatory cytokines, leading to escalated cell growth and reduced cell death (16). As well, proinflammatory cytokines, such as tumor necrosis factor ␣ (TNF␣) and interleukin-1 (IL-1), increase expression of GR␤ via the NF-B pathway (17). Ascending levels of GR␤ in asthma patients cause many disease complications, including GC resistance that can verge to a complete loss of drug response (18). Importantly, both leukemia (19) and systemic lupus erythematosus (20) have been linked to high levels of GR␤, which may underlie the exacerbated immune cell growth observed in these patients. How GR␤ regulates growth is unknown, but the mechanism may involve inhibition of GR␣-induced apoptosis or direct GR␤ epigenetic signaling. A previous study reported gene-specific activity for GR␤ that is independent of GR␣ (21), a known inhibitor of growth. Therefore, in this study, we investigated the effect of GR␤ on the insulin signaling pathways guiding proliferation. Our findings demonstrate that GR␤ plays an important role in the control of insulin signaling by suppressing PTEN, resulting in the enhancement of growth.

EXPERIMENTAL PROCEDURES
Cell Lines and Culture-The mouse 3T3-L1, C2C12, RAW 264.7, S49 and embryonic fibroblast (MEF) were routinely cultured and maintained in Dulbecco's Modified Eagles's Medium (DMEM) containing 10% bovine calf serum or FBS with 1% penicillin-streptomycin. The scramble and GR␤ MEF cell lines were grown as previously described (11).
Whole Cell Extraction-Cells were washed and collected in 1ϫ PBS followed by centrifugation at 1500 ϫ g for 10 min. The supernatant was discarded, and the pellet was resuspended in 1ϫ PBS. After a short spin at 20,800 ϫ g for 5 min at 4°C, the pellet was rapidly frozen on a dry ice ethanol mix and stored at Ϫ80°C for 30 min. The frozen pellet was then resuspended in 3 volumes of cold whole cell extract buffer (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, pH 7.4) with protease inhibitors and incubated on ice for 10 min. The samples were centrifuged at 100,000 ϫ g for 5 min at 4°C. Protein levels were measured spectrophotometrically by a Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE). The supernatants were either stored at Ϫ80°C or used immediately for Western analysis to determine protein expression levels.
Quantitative Real-Time PCR Analysis-Total RNA was extracted from mouse tissues using 5-Prime PerfectPure RNA Cell Kit (Fisher Scientific Company, LLC). Total RNA was read on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR amplification of the cDNA was performed by quantitative real-time PCR using TrueAmp SYBR Green qPCR SuperMix (Smart Bioscience). The thermocycling protocol consisted of 10 min at 95°C, 40 cycles of 15 s at 95°C, 30 s at 60°C, and 20 s at 72°C and finished with a melting curve rang-ing from 60 -95°C to allow distinction of specific products. Primer sequences were downloaded from a primer database. Normalization was performed in separate reactions with primers to GAPDH.
Generation of Lentiviral Constructs-To establish a 3T3-L1 cell line that has mGR␤ stably overexpressed, mGR␤ cDNA was ligated into the XbaI/XhoI sites of the FG12 vector that has an independent GFP marker and transformed in DH5␣ cells (Invitrogen). The construct was co-transfected together with vectors expressing gag-pol, REV, and VSV-G into 293FT cells (Invitrogen) to generate a third generation lentiviral construct. Transfection was achieved using GeneFect (Alkali Scientific, Inc.) using 100 ng of total DNA per cm 2 of the growth plate or well. The supernatants were harvested, and the cell debris was removed by centrifugation at 2000 ϫ g. The supernatant was used to infect 3T3-L1 cells after addition of polybrene (5 ng/ml, Sigma) to establish cell lines with stable overexpression of mGR␤ mRNA (3T3-GR␤) or expressing empty vector (3T3-V). After 72 h, the cells were sorted by flow cytometry for GFP by the Flow Cytometry Core Facility at the University of Toledo Health Science Campus. GFP-positive cells were used for all experiments.
Transient Transfection-For transient transfection cells were plated on a 6-well dish in DMEM containing 10% calf serum prior to transfection and allowed to grow to 85-90% confluency. Cells were washed with OPTI-MEM and transfected using GeneFect (Alkali Scientific, Inc.), according to the manufacturer's protocol. OPTI-MEM was removed after 5 h and DMEM containing 10% calf serum was added. All insulin treatments were done 24 h post-transfection for 30 min.
Targeting of Mouse GR␤ mRNA-Targeting of mouse GR␤ was designed and purchased using the Integrated DNA Technologies (IDT) sciTools RNAi Design website. Two siRNA targets were designed: Seq 1 (gatgtaagtaccaaacataaatc) and Seq 2 (gtcagagatacgtaagagatccta), as well as scramble control. Transient transfection of the siRNAs was performed using GeneFect (Alkali Scientific, Inc.) for 24 h.
Targeting of the Mouse GR Gene-A peptide nucleic acid (PNA) targeting the branch point sequences (BPS) in intron 8 of the mouse GR gene (previously identified in Ref. 11) was designed using PANAGENE. Two different PNAs were designed: CPP-PNA-i8BPS (gactgattggtatat) and CPP-PNA-i8C (atataccaatcagtc). All PNAs were attached to an O Linker and a modified TAT protein (VQRKRQKLMP) for delivery into the cell (cell penetrating peptide -CPP). Treatment with CPP-PNAs was performed for 24 h.
Promoter Reporter Assays-Expression vector for mGR␤ (pMGR␤-H57) was constructed as previously described (11). PTEN promoter (full-length, truncations and mutants) activity was measured by luciferase, and these constructs were made as defined in Ref. 22, and pRL-CMV Renilla reporter for normalization to transfection efficiency. Transient transfection was achieved using GeneFect (Alkali Scientific, Inc.). 24-h posttransfected cells were lysed, and the luciferase assay was performed using the Promega luciferase assay system (Promega).
Gel Electrophoresis and Western Blotting-Whole cell extracts (WCE) were prepared by freezing the cell pellet overnight at Ϫ80°C. The pellet was then resuspended in 3 volumes of WCE buffer (20 mM HEPES, 0.42 M NaCl, 0.2 M EDTA, 25% glycerol, pH 7.4) plus protease inhibitor mixture and incubated on ice for 10 min followed by 100,000 ϫ g centrifugation at 4°C. Protein samples were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-FL membranes. Membranes were blocked at room temperature for 1 h in TBS (TBS; 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl) containing 3% BSA. Subsequently, the membrane was incubated overnight at 4°C with FiGR antibody for mGR␣ (Santa Cruz Biotechnology, Dallas, Texas) or rMGR␤ antibody for mGR␤ (described in Ref. 11) at a dilution of 1:1000 in TBS. Antibodies that recognize HSP90, Akt1, Akt2, pAkt, and total Akt were purchased from Santa Cruz Biotechnology and added at a dilution of 1:1000 in TBS. After three washes in TBST (TBS plus 0.1% Tween 20), the membrane was incubated with an infrared anti-rabbit (IRDye 800, green) or anti-mouse (IRDye 680, red) secondary antibody labeled with IRDye infrared dye (LI-COR Biosciences) (1:15,000 dilution in TBS) for 2 h at 4°C. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LI-COR Biosciences).
Proliferation Assays-Scramble and mGR␤ OE 3T3-L1 cells (1 ϫ 10 4 cells per well) were plated in 24-well plates in DMEM containing 10% calf serum. The growth rate was determined as a function of time for 0 -5 days. To determine the effect of insulin-stimulated cell growth, Scramble and mGR␤ OE 3T3-L1 cells were grown in DMEM containing dialyzed serum were treated with 100 nM insulin for 24 h, cell proliferation was determined by a calorimetric assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazoline bromide) as previously described (23).
Statistical Analysis-Data were analyzed with Prism 5 (GraphPad Software, San Diego, CA) using analysis of variance combined with Tukey's post-test to compare pairs of group means or unpaired t tests. Additionally, two-way ANOVA was utilized in multiple comparisons, and followed by the Bonferroni post hoc analysis to identify interactions. p values of 0.05 or smaller were considered statistically significant.

Detection of GR␤ Expression in Tissues and Cells from Different Murine Lineages-
The recent discovery of the mouse GR␤ (mGR␤) has opened new avenues for studying glucocorticoids and the actions of glucocorticoid receptors (11): whether mouse GR␤ (mGR␤) is expressed by various cell types and lineages are largely unknown. To detect mGR␤ and mGR␣ in different tissues of murine origin, we analyzed lysates from muscle, liver, spleen, kidney, and heart (Fig. 1A). The mGR␣ was detected at 97 kDa in muscle, liver, spleen, and to a lesser extent in kidney and heart. We detected the GR␤ protein in muscle and liver at 92 and 102 kDa. In spleen, kidney, and heart only the 102-kDa GR␤ band was present. The shift in molecular weight was also noticed for human GR␤ in heart and skeletal muscle tissues (24). We therefore wanted to analyze the expression of mGR␤ in murine cells from hepatic and muscle lineages (Fig.  1B). We found in Hepa1c1c7 hepatocytes that mouse GR␤ is detectable at the 92 and 102 kDa bands, and C2C12 myocytes had a single band at 102 kDa. The mGR␣ levels were lower in C2C12 myocytes compared with Hepa1c1c7 hepatocytes. These results suggest that cells from mesenchymal and endodermal origins may have a differential GR␤ and GR␣ expression. To compare murine cells of mesenchymal origin we used mouse embryonic fibroblast (MEF), 3T3-L1 and C2C12 cells (Fig. 1C). Western blotting analysis showed that MEF and C2C12 have similar detectable levels of mGR␤ (p ϭ 0.23). C2C12 cells have reduced mGR␣ levels compared with MEF (p ϭ 0.007). Interestingly, the 3T3-L1 cell line has very low to undetectable levels of mGR␤ and mGR␣. Furthermore, we determined mGR␤ and mGR␣ expression in two murine immune cell lines, RAW 264.7 leukemic monocyte/macrophage, and S49 lymphoma cells (Fig. 1D). Interestingly, S49 lymphocytes have significantly higher expression of mGR␤ (p ϭ 0.0036) compared with RAW 264.7 cells, and lower expression of mGR␣ (p Ͻ 0.001).
Mouse GR␤ Sensitizes to Insulin Signaling-We have previously shown that mGR␤ mRNA was increased in MEF cells and in livers of mice that were fasted and refed (11). Here, we treated MEF cells with 100 nM insulin for 24 h and determined mGR␤ expression. Confirming our previous mRNA results, mGR␤ protein expression was significantly increased following insulin treatment and no change was observed in mGR␣ ( Fig. 2A). The capacity of GR␤ in insulin signaling is unknown. Therefore, we determined the impact of mGR␤ on the phosphorylation of Akt with and without insulin treatments for 30 min in MEF cells overexpressing mGR␤. To our surprise, overexpression of mGR␤ basally increased pAkt, which was further elevated upon insulin treatment compared with vector controls (Fig. 2B). Fig. 1C showed that 3T3-L1 cells have undetectable levels of mGR␤ and mGR␣ protein. Therefore, we created a cell line that stably overexpressed only mGR␤ (3T3-GR␤) or vector control (3T3-V) to elucidate the actions of mGR␤ outside of its known GR␣ inhibitory role (11,25). In Fig. 3A, we show that the 3T3-GR␤ cell line highly expressed mGR␤ protein. Interestingly, this cell line had significantly reduced PTEN expression  (p ϭ 0.0001), suggesting a positive role of mGR␤ in insulin signaling. Therefore, we measured protein expression of Akt1 and Akt2. Unexpectedly, the 3T3-GR␤ cells had significantly higher Akt1 (p ϭ 0.0060) with no change in Akt2 (p ϭ 0.1867) expression (Fig. 3A). To confirm that mGR␤ regulates phosphorylation of Akt, we treated 3T3-GR␤ and 3T3-V cells with insulin for 30 min. Basally, mGR␤ significantly increased pAkt, which was further enhanced upon insulin treatments (Fig. 3B). Because mGR␤ inhibited expression of PTEN and enhanced Akt1, which has been shown to regulate cellular proliferation (26), we investigated proliferation in the 3T3-GR␤ cells (Fig.  3C). Growth of 3T3-GR␤ cells was significantly higher (ANOVA, p ϭ 0.0001) than the 3T3-V cells (Fig. 3D), specifically at days 4 (p Ͻ 0.001) and 5 (p Ͻ 0.001) of growth. Additionally, 24 h of insulin treatment significantly increased growth of 3T3-GR␤ cells (p ϭ 0.0405).

Mouse GR␤ Enhances Growth by Suppression of PTEN-Our studies in
Targeting of Mouse GR␤ Enhances PTEN Expression-We designed two siRNAs targeting mGR␤ expression to determine if PTEN expression is enhanced with reduced levels of mGR␤ (Fig. 4A). Sequence 1 (Seq 1) was designed within the coding region of mGR␤ within intron 8. Seq 2, however, was designed outside of the coding region of mGR␤ to determine if mGR␤ expression can be regulated outside this region. Seq 1 significantly decreased mGR␤ expression by 62%, resulting in 2.1-fold enhancement of PTEN expression (p ϭ 0.0113) (Fig. 4B). Seq 1 targeting of mGR␤ had no effect on mGR␣ expression. Interestingly, Seq 2 had no effect on mGR␤ expression demonstrating that siRNA targeting of mGR␤ must occur within the coding region of intron 8 of the mouse glucocorticoid receptor gene.
Targeting of the Mouse GR Gene Enhances mGR␤ Expression-We have previously identified two putative branch point sequences (BPS) within intron 8 of the mouse GR gene (11). We wanted to determine if specific targeting of the BPS utilizing a PNA conjugated to a cell-penetrating peptide (CPP) (modified TAT protein) can bind to intron 8 of the mouse GR gene to regulate alternative splicing of GR␤ or GR␣. A dose-dependence treatment with CPP-PNA-i8BPS significantly enhanced mGR␤ expression at 100 nM and 500 nM, and decreased expression of mGR␣ (Fig. 5A). Treatment with the control CPP-PNA-i8C resulted in no change in either mGR␤ or mGR␣ mRNA expression. We have shown in Fig. 3A that increasing mGR␤ expression decreased PTEN. Therefore, we measured mRNA expression of PTEN with 100 nM BPS1-CPP-i8BPS treatment for 24 h. Increasing mGR␤ expression via CPP-PNA-i8BPS decreased PTEN mRNA expression by 38% (Fig. 5B). In Fig. 5C, immunoblotting confirmed that treatment with 100 nM of BPS1-CPP-i8BPS significantly decreased expression of PTEN (p ϭ 0.0405), enhanced mGR␤ (p ϭ 0.0358), and no change in GR␣ (p ϭ 0.3716).
Mouse GR␤ Regulates Promoter Activity of PTEN-To determine if mGR␤ inhibits the PTEN promoter we utilized the PTEN-luciferase construct. Overexpression of mGR␤ in MEF cells resulted in a significant (p ϭ 0.0136) reduction of PTEN promoter activity by 21% compared with vector controls (Fig.  6A). In the 3T3-L1 cells, which have very low levels of both GR␤ and GR␣ (Fig. 1A), overexpression of GR␤ resulted in a robust 56% reduction in PTEN promoter activity (Fig. 6A). We determined if targeting of PTEN expression would have a reciprocal effect on mGR␤ or mGR␣ expression. Interestingly, siRNA knockdown of PTEN in MEF cells had no effect on mGR␤, mGR␣ or Akt1 mRNA expression (Fig. 6B). In the mGR␤ shRNA knockdown MEF cells published in (11) (Fig. 6C), PTEN mRNA expression was significantly increased (p Ͻ 0.05) with no change in Akt1 (Fig. 6D). Insulin treatment in 3T3-L1 and MEF cells increased PTEN promoter activity, which was inhibited by overexpression of GR␤ (Fig. 6E). Truncations of the PTEN promoter showed that GR␤ is targeting the Ϫ1344 to Ϫ1001 region (Fig. 7A). Mutations of the PTEN promoter in the Ϫ1228 to Ϫ1149 region showed that GR␤ is specifically targeting the SubB and SubC mutant regions (Fig. 7B).

DISCUSSION
This is the first study to demonstrate that GR␤ can enhance proliferation by the suppression of PTEN. Here we report that GR␤ protein expression is elevated by insulin treatment, which enhances growth through modulating PTEN promoter activity. Increasing levels of PTEN has been shown to be essential for the suppression of PI3-kinase induced tumor growth in mice (27). Insulin increased PTEN promoter activity over a 24-h period, which implies a negative feedback regulation. Interestingly, GR␤ suppressed the negative feedback regulation decreasing PTEN expression, leading to exacerbated Akt phosphorylation and growth. This suggests that long term increases in GR␤ expression may enhance growth by suppressing PTEN leading to over stimulation of PI3-kinase and Akt. Inhibitors of the PI3-kinase pathway enhanced GC induced apoptosis in human lymphoma cells (28), which indicate a positive feedback between glucocorticoids/GR␣ and PTEN in the suppression of growth. The interplay between GR␣ and PTEN was demonstrated in T-cells extracted from human leukemia patients, which had increased PTEN expression following GC treatment (29). However, patients that relapsed had a complete loss of PTEN expression. The recruitment of GR␤ to the PTEN promoter uncovers an essential pathway for understanding the mechanism of the inhibition of GC induced apoptosis and sensitization to Akt stimulated growth. The GR␤ inhibition of GCinduced growth has been shown in peripheral mononuclear cells, in which cytokine stimulation increased GR␤ expression and prevented apoptosis (30). Interestingly, we show that protein expression of GR␤ is more abundant in mouse lymphoma cells compared with GR␣. Similarly, high expression levels of GR␤ have been reported in immune cells of lupus (20) and leukemia (19) patients, suggesting that a leading target for inhibiting growth in these cells would be GR␤. Here we show that siRNA targeting of GR␤ in MEF cells concomitantly increased PTEN expression.
We have also discovered that GR␤ selectively increases Akt1 expression over that of Akt2. Our finding of GR␤ in mice (11) and the recent report by Dubois et al. on GR␤ in the rat (31), suggest that rodent GR␤ has a possible role in metabolic signaling (11). Therefore, we determined the impact of elevated GR␤ on the insulin signaling pathway, showing that it leads to exacerbated Akt phosphorylation and growth. Akt exists as three structurally similar, yet different, isoforms: Akt1, Akt2, and Akt3. The functionality of these isoforms has been shown to be divergent, even though their structures are similar. The Akt1 isoform has been shown to be essential in the regulation of development and growth (26,32). In contrast, the Akt2 isoform is mostly involved in glucose uptake (33) and adipogenesis (34). These actions have lead several investigators to study the involvement of Akt2 in metabolic diseases, such as diabetes and obesity (33). Akt3 has been the least studied of the isoforms, but investigations show that Akt3 may have a possible role in brain development (35). The growth regulation properties of Akt1 have made it a major target in cancer studies (32,36,37), especially PTEN-mediated inhibition of Akt1 in tumor development (38). The selectively of GR␤ for Akt1 indicates a potential capacity of this nuclear receptor in growth pathways. This was demonstrated by lentiviral overexpression of GR␤ in 3T3-L1 cells, which resulted in elevated Akt1 expression and enhanced insulin-stimulated proliferation. Akt1 is a direct target of the insulin/PI3-kinase induced growth pathway, and deletions have been beneficial in cancer, especially in lung tumorigenesis by mutant K-ras (36). Additionally, the suppression of the PI3kinase/Akt1 growth pathway by gene targeting or by the PI3-kinase inhibition has been shown to decrease tumor mass size (37). Interestingly, Piovan et al. identify Akt1 as a negative regulator of GR␣ by phosphorylating serine 134, which resulted in GC resistance in T cell acute lymphoblastic leukemia (39). They also show that suppression of PTEN inhibits GR␣ expression and activity, enhances Akt phosphorylation, resulting in GCresistance.
To date, most studies have only considered GR␤ as an inhibitor of GC action, primarily due to its ability to heterodimerize with GR␣ (18,40) and to recruit histone deacetylases inhibiting gene activity (41). The impedimentary action of GR␤ on GR␣ was recently demonstrated by Varricchio et al. in polycythemia vera (PV) patients, which have increased erythroid cell growth (42). This study found that PV patients have a polymorphism in the human GR gene that leads to the stabilization of the GR␤ mRNA, resulting in higher protein levels. Interestingly, these patients have exacerbated GR␤/GR␣ heterodimerization and expansion of erythroblast. GR␣ is known to heterodimerize with other members of the steroid receptor family, such as the mineralocorticoid (MR) and androgen (AR) receptors (43,44). It is also possible that GR␤ may regulate steroid receptor activity other than GR␣. The GR␤/Akt1 growth axis may have an important function in the regulation of AR activity. Knockdown of hGR␤ by siRNA reduced androgen induced growth in LNCaP prostate cancer cells (45). However, Akt1 and PTEN were never investigated, and it is only now coming to light that GR␤ has a consummate role in these pathways. Furthermore, bombesin treatment attenuated GC-induced apoptosis in PC-3 human prostate cancer cells by increasing hGR␤ expression (46), albeit, through GR␤ inhibition of PTEN. Importantly, GR␤ may have a major function in the development of several cancers. The HT-29 colon cancer and MCF-7 breast carcinoma (47), as well as, Hut-78 T-and Raji B-lymphoma cell lines (48) have shown high expression of GR␤. Interestingly, treatment with growth inhibitors 5-aza-2Ј-deoxycytidine (5-dAzaC), sodium butyrate (NaBu), and trichostatin A (TSA) reduced GR␤ and increased GR␣ expression in all of these cancer cell lines (47,48). Unfortunately, sensitivity to GC-induced apoptosis or expression of PTEN was never tested in these studies. Likewise, hepatocarcinoma (HepG2) and osteosarcoma cells (SaOS-2) have elevated expression of GR␤, which was found to be mostly located within the nucleoli (49,50).
In conclusion, the predominant aspects studied for GR␤ have been on the inhibition of GR␣. Our studies clearly show that GR␤ has a preeminent role in insulin signaling and growth. These properties have not been previously known for GR␤, especially the epigenetic Akt1/PTEN growth signaling that is independent of GR␣ (Fig. 8). Given that Akt1 regulates embryonic and fetal growth (26), this work suggests that GR␤ may have a paramount role in development and proliferation. Thus, the GR␤/Akt1 axis is emerging as a major signaling paradigm regulating growth, which may also lead to GC-resistance in cancer. Therapeutics inhibiting GR␤ may increase sensitivity to GCs, as well as increase PTEN expression allowing for the regulation of growth.