cAMP-stimulated transcription of DGKθ requires steroidogenic factor 1 and sterol regulatory element binding protein 1.

Diacylglycerol kinase (DGK)θ is a lipid kinase that phosphorylates diacylglycerol to form phosphatidic acid (PA). We have previously shown that PA is a ligand for the nuclear receptor steroidogenic factor 1 (SF1) and that cAMP-stimulated expression of SF1 target genes requires DGKθ. In this study, we sought to investigate the role of cAMP signaling in regulating DGKθ gene expression. Real time RT-PCR and Western blot analysis revealed that dibutyryl cAMP (Bt2cAMP) increased the mRNA and protein expression, respectively, of DGKθ in H295R human adrenocortical cells. SF1 and sterol regulatory element binding protein 1 (SREBP1) increased the transcriptional activity of a reporter plasmid containing 1.5 kb of the DGKθ promoter fused to the luciferase gene. Mutation of putative cAMP responsive sequences abolished SF1- and SREBP-dependent DGKθ reporter gene activation. Consistent with this finding, chromatin immunoprecipitation assay demonstrated that Bt2cAMP signaling increased the recruitment of SF1 and SREBP1 to the DGKθ promoter. Coimmunoprecipitation assay revealed that SF1 and SREBP1 interact, suggesting that the two transcription factors form a complex on the DGKθ promoter. Finally, silencing SF1 and SREBP1 abolished cAMP-stimulated DGKθ expression. Taken together, we demonstrate that SF1 and SREBP1 activate DGKθ transcription in a cAMP-dependent manner in human adrenocortical cells.

. DGK -null mice exhibit several neural abnormalities, including a higher resistance of electroconvulsive shock ( 5 ) and increased cyclooxygenase 2 and tyrosine hydroxylase expression ( 6 ), suggesting a role for DGK in regulating synaptic activity. Mice lacking DGK ␣ ( 7 ) or DGK ( 8 ) exhibit enhanced T cell function and demonstrate a role for these kinases in controlling DAG metabolism during the immune response. DGK isoforms have been implicated in various other cellular processes including inhibition of Rap1 signaling ( 9 ) and retinoblastoma-mediated cell cycle control ( 10 ). DGK is activated by nerve growth factor in PC12 cells ( 11 ) and thrombin in IIC9 fi broblasts ( 12,13 ), whereas DGK promotes myogenesis in C2C12 cells ( 14 ) and DGK ␥ plays a role in regulating the cell cycle in CHO-K cells ( 15 ).
To date, 10 mammalian DGKs have been identifi ed that are divided into fi ve groups based on functional domains ( 16,17 ). However, all isoforms contain cysteine-rich zinc fi nger-like structures, a conserved catalytic region (18)(19)(20)(21). DGK , the sole member of group V, is comprised of three cysteine-rich domains (CRDs), a proline/glycine-rich domain at its N terminus, and a pleckstrin homology (PH) with an overlapping Ras-binding domain ( 22 ). While the functions of many of the other domains in DGK are unclear, the catalytic activity requires all domains of the enzyme ( 23 ). It has been postulated that the CRDs of the enzyme are required both for correct folding of the protein and for substrate presentation ( 23 ). Mutation of the CRD of DGK diminishes DAG-induced translocation of the enzyme to the plasma membrane ( 24 ); whereas the interaction between DGK and the nuclear receptor steroidogenic factor 1 (SF1) requires the PH domain ( 25 ).
Abstract Diacylglycerol kinase (DGK) is a lipid kinase that phosphorylates diacylglycerol to form phosphatidic acid (PA). We have previously shown that PA is a ligand for the nuclear receptor steroidogenic factor 1 (SF1) and that cAMPstimulated expression of SF1 target genes requires DGK . In this study, we sought to investigate the role of cAMP signaling in regulating DGK gene expression. Real time RT-PCR and Western blot analysis revealed that dibutyryl cAMP (Bt 2 cAMP) increased the mRNA and protein expression, respectively, of DGK in H295R human adrenocortical cells. SF1 and sterol regulatory element binding protein 1 (SREBP1) increased the transcriptional activity of a reporter plasmid containing 1.5 kb of the DGK promoter fused to the luciferase gene. Mutation of putative cAMP responsive sequences abolished SF1-and SREBP-dependent DGK reporter gene activation. Consistent with this fi nding, chromatin immunoprecipitation assay demonstrated that Bt 2 cAMP signaling increased the recruitment of SF1 and SREBP1 to the DGK promoter. Coimmunoprecipitation assay revealed that SF1 and SREBP1 interact, suggesting that the two transcription factors form a complex on the DGK promoter . Finally, silencing SF1 and SREBP1 abolished cAMP-stimulated DGK expression. Taken together, we demonstrate that SF1 and SREBP1 activate DGK transcription in a cAMPdependent manner in human adrenocortical cells. - Supplementary key words diacylglycerol kinase • adrenal cortex • cAMP Diacylglycerol kinases (DGKs) are intracellular lipid kinases that phosphorylate diacylglycerol (DAG) to form phosphatidic acid (PA), which is linked to lipid metabolism and signaling (1)(2)(3). For example, targeted disruption of DGK ␦ in mice impairs epidermal growth factor receptor expression and increases protein kinase C (PKC) activity with 10% fetal bovine serum (Mediatech, Inc.), antibiotics, and antimycotics. SF1 and sterol regulatory element binding protein 1 (SREBP1) knockdown cell lines were generated by transfecting H295R cells with short hairpin RNA (shRNA) plasmids (in the pGFP-V-RS HuSH vector; Origene, Rockville, MD) containing the following oligonucleotides: SF1 5 ′ -TCC TGG CCG TGC  CAT CAA GTC TGA GTA CC and SREBP1 5 ′ -ATC TAT GTG  GCG GCT GCA TTG AGA GTG AA. Stable clones were selected using 10 g/ml puromycin (Mediatech, Inc.). H295R cells expressing tetracycline-inducible DGK shRNA were generated using the BLOCK-iT Inducible H1 RNAi Entry Vector Kit (Invitrogen) as previously described ( 37 ). To construct an inducible vector for DGK shRNA, the following sequences were cloned into pENTR/H1/TO: 5 ′ -ACC GCC CAG TAT TGA AGG CCT CAT CTT CAC GAA TGA AGA TGA GGC CTT CAA TAC TGG G-3 ′ and 5 ′ -AAA CCC AGT ATT GAA GGC CTC ATC TTC ATT CGT GAA GAT GAG GCC TTC AAT ACT GGG C-3 ′ . H295R-TetR cells were stably transfected with the constructed pENTR/H1/TO-DGK shRNA expression vector or the control vector using GeneJuice (EMD Biosciences), and cell clones were selected using 50 g/ml zeocin. Clones were treated with 5 g/ml tetracycline for 96 h and suppression of DGK protein levels in each clone was confi rmed by Western blotting using an anti-DGK antibody (Sigma).
The ability of distinct isoforms to exert regulatory control occurs through unique interactions with protein partners, and differential subcellular localization of DGK isoforms is thought to enable local regulation of DAG and PA concentrations for spatial and temporally separated cellular processes. Many studies have demonstrated roles for compartmentalized DGK activity in nuclear processes. Both DGK ( 26 ) and DGK ( 14 ) are localized in punctate structures that are enriched in pre-mRNA splicing factors called nuclear speckles. DGK is colocalized with hyperphosphorylated RNA polymerase II and the splicing factor SC-35 in the nuclear speckles of various cell types, including PC12, HeLa, and MCF-7 ( 26 ). Interestingly, nuclear speckles have been shown to sequester posttranslationally modifi ed SF1 ( 27,28 ). SF1 induces the transcription of genes involved in steroid hormone biosynthesis and endocrine development and function (29)(30)(31). We have previously shown that cAMP signaling increases the transcription of CYP17A1 by stimulating the binding of SF1 to the CYP17A1 promoter ( 32,33 ). We have also shown that DGK regulates the production of PA, a ligand for SF1 that is produced in response to cAMP signaling ( 25 ). DGK acts as a coregulatory protein by binding to SF1 when the receptor is bound to chromatin. The PA produced in response to DGK activation stimulates SF1-dependent gene transcription by promoting coactivator recruitment to SF1 target genes, thereby inducing the mRNA expression of CYP17A1 and several other steroidogenic genes. In contrast, inhibition of DGK activity attenuates the binding of SF1-dependent gene expression, and silencing the expression of DGK expression inhibits cAMP-dependent CYP17A1 transcription. Finally, we have also shown that LXXLL motifs in DGK mediate a direct interaction of SF1 with the kinase and may facilitate ligand delivery ( 25 ). To date, studies have demonstrated that DGK is regulated by intracellular targeting ( 24 ), membrane lipids ( 12 ), protein-protein interactions ( 34 ), and intrinsic activity ( 12 ). However, the factors that control DGK gene expression in the adrenal cortex are poorly understood. In this study, we defi ned the role of cAMP signaling in regulating the expression of DGK .

Materials
Dibutyryl cAMP (Bt 2 cAMP) and tetracycline were obtained from Sigma (St. Louis, MO) and H89 from EMD Biosciences (La Jolla, CA).

RNA isolation and quantitative RT-PCR
Cells were sub-cultured onto 12-well plates and 24 h later treated with 0.4 mM Bt 2 cAMP for 1-24 h. Total RNA was extracted using Iso-RNA Lysis Reagent (5 Prime Inc., Gaithersburg, MD) and amplifi ed using a One-Step SYBR Green RT-PCR Kit (Thermo Fisher Scientifi c Inc., Waltham, MA) and the primer pairs described in Li et al. ( 25 ). DGK expression was normalized to ␤ -actin content and calculated using delta-delta cycle threshold ( ⌬ ⌬ CT) method.

Nuclear and cytoplasmic extract isolation
H295R cells were cultured in 100 mm dishes and treated with Bt 2 cAMP for 48 h. Cytoplasmic and nuclear extracts were harvested from H295R cells and separated using Thermo NE-PER ® nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL). Western blotting analysis was carried out as described above. Blots were probed with anti-DGK (1:1000, HPA026797; Sigma) and anti-lamin A/C (1:5000, sc-376248; Santa Cruz Biotechnology) or anti-␤ -tubulin (1:2000, sc-23949; Santa Cruz Biotechnology).

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) assays were performed as described previously in ( 32,39,40 ). Briefl y, H295R cells were sub-cultured onto 150 mm dishes and treated with Bt 2 cAMP for 60 min. Cells were treated with 1% formaldehyde for 10 min at room temperature and then incubated for 5 min with 0.125 M glycine. After twice washing with PBS, cells were harvested into RIPA after 24 h treatment with Bt 2 cAMP ( Fig. 1A ), but had no effect on the mRNA expression of other DGK isoforms ( Fig. 1A ). Next, we assessed the kinetics of the DGK response to Bt 2 cAMP by treating H295R cells for 1-24 h. The results revealed that Bt 2 cAMP activation rapidly increased DGK mRNA expression by 1.5-fold at the 3 h time point with a maximal 3.7-fold increase in DGK mRNA expression occurring at the 21 h time point ( Fig. 1B ). Consistent with an increase in mRNA expression, Bt 2 cAMP treatment led to an increase in DGK protein expression by 2.2-and 2.7-fold after 48 h and 72 h treatment, respectively ( Fig. 1C ).

Effect of kinase on cAMP-stimulated DGK mRNA expression
In the human adrenal cortex, the action of cAMP is mediated by the cAMP-dependent protein kinase A (PKA) ( 41 ). To determine if Bt 2 cAMP stimulated DGK expression required PKA, H295R cells were treated with H89 or the mitogen-activated protein kinase inhibitor U0126 . As shown in Fig. 2 , H89 treatment attenuated the cAMP activation on DGK mRNA expression. Conversely, no significant effect was observed with U0126.

cAMP increases DGK reporter gene activity
Next we sought to defi ne the mechanism by which cAMP stimulation induces DGK expression and cloned 1.5 kb of the DGK promoter into a reporter gene plasmid fused to the Firefl y luciferase gene and transfected the construct into H295R cells. As shown in Fig. 3A , Bt 2 cAMP treatment signifi cantly increased the transcriptional activity of the 1.5 kb reporter gene by 1.9-fold. In silico analysis of the DGK promoter revealed several putative SF1 binding sites ( Fig. 3B ). Notably, one of these putative SF1 binding sites overlapped with response elements for the SREBP family. SREBPs are a family of basic helix-loop-helix leucine zipper buffer. The purifi ed chromatin solutions were immunoprecipitated using 5 g of anti-acetyl-K5, K8, K12, K16 histone H4 (06-866; Millipore, Temecula, CA), anti-SF1 (07-618; Millipore), SREBP1 (sc-8984; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-IgG protein A/G plus (sc-2003; Santa Cruz Biotechnology). Realtime PCR was carried out using the following primer sets: forward 5 ′ -CAG AGT CCA CAG CCC CCA GCC CCT TTC AGG and reverse 5 ′ -CTG CCT CGT GCG CGC CAC GGG TCT TGT TCA. Output DNA (immunoprecipitated promoter region) was normalized to input DNA. PCR products were separated on 2% agarose gels and the EtBr-stained bands imaged using a VersaDoc 4000 (Bio-Rad).

Coimmunoprecipitation
CV1 cells were plated onto 100 mm dishes and transfected with pCMV6-GFP-SF1, pcDNA 3.1-FLAG SREBP1c for 48 h. Five percent of lysates were retained as input and the remaining cell lysates were incubated with an anti-FLAG M2 mouse monoclonal antibody (5 g; F1804, Sigma) and protein A/G agarose beads (Santa Cruz Biotechnology) overnight at 4°C with rotation. Beads were washed three times with RIPA buffer and twice with PBS and the immobilized proteins separated by SDS-PAGE. Output blots were probed with anti-SF1 (1:5000, 07-618; Millipore) and input blots with anti-FLAG (1:2500, F1804, Sigma). Expression was detected using an ECF Western blotting kit (GE Biosciences) and visualized using a VersaDoc 4000 imager (Bio-Rad).

PA assay
H295R cells were grown on 100 mm dishes and then treated with Bt 2 cAMP from 24 h to 48 h and total lipid extract was harvested. Nuclei were purifi ed using a Nuclei Pure kit (Sigma) and PA content was quantifi ed using a Total PA kit (Cayman, Ann Arbor, MI) on a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA) at an excitation wavelength of 530-540 nM and an emission wavelength of 585-595 nm. Data was quantifi ed using through SoftMax Pro software (Molecular Devices).

DAG assay
H295R cells were cultured onto 6-well plates and treated with Bt 2 cAMP from 72 h and cells were harvested with PBS. PBS was aspirated and the content of DAG in each sample was determined using a Human DAG ELISA kit (MyBioSource, Inc., San Diego, CA).

Statistical analysis
One-way ANOVA and Tukey-Kramer multiple comparisons were performed using Prism 5.0 (GraphPad Software, San Diego, CA). Signifi cant difference value was set as P < 0.05.

cAMP induces DGK mRNA expression
We have previously shown that adrenocorticotropic hormone (ACTH) signaling rapidly increases DGK activity ( 25 ). Therefore, in this study we sought to determine the effect of increased intracellular cAMP on DGK gene expression. H295R human adrenocortical cells were treated with Bt 2 cAMP for 24 h and RNA isolated for qRT-PCR. DGK mRNA expression was increased by 2.9-fold To determine the effect of SF1 and SREBP1c on DGK reporter gene transcription, we transfected expression plasmids for these transcription factors in H295R cells and quantifi ed luciferase activity. Consistent with the Bt 2 cAMP effect, cotransfection with an SF1 expression and SREBP1c plasmids resulted in a 1.8-and 2.7-fold increase in DGK reporter gene activity, respectively. Moreover, overexpression of both transcription factors resulted in a 4.2-fold increase in DGK luciferase activity, with Bt 2 cAMP further stimulating DGK reporter gene transcription. Transfection of DGK reporter gene plasmids harboring mutations at putative SF1/SREBP1c sites ( Fig. 3B ) revealed that mutation of region M5 ( Ϫ 775/ Ϫ 776) had no signifi cant effect on basal DGK promoter reporter gene activity, whereas (bHLHLZ) transcription factors that regulate fatty acid, triglyceride, and cholesterol metabolism (42)(43)(44). In contrast to other bHLHLZ transcription factors, SREBPs bind to both E-boxes (5 ′ -CANNTG-3 ′ ) and sterol regulatory element (SRE) sequences (5 ′ -TCACNCCAC-3 ′ ) ( 45 ). There are three isoforms of SREBPs in mammals: SREBP1a, SREBP1c, and SREBP2. However, because SREBP1s are more specifi c for activation of fatty acid synthesis and SREBP1c is the predominant isoform in murine and human tissues such as liver, adrenal gland, and brain ( 46 ), whereas SREBP2 is more selective for regulating cholesterol production ( 42 ), we focused on SREBP1c. Further, our previous work has shown that sphingosine-1-phosphatestimulated CYP17A1 transcription requires SREBP1c ( 47 ). Luciferase activity in lysates isolated from control ( Ϫ ) and Bt 2 cAMP-treated cells (+) was quantifi ed by luminometry. Data are expressed as the fold change in pGL3-DGK (Firefl y luciferase) reporter gene activity over the untreated control group mean, are normalized to pRL-CMV (Renilla luciferase) activity and represent the mean ± SEM of three separate experiments, each performed in triplicate. Asterisks (*) and carats (^) indicate a statistically signifi cant difference ( P < 0.05) from the pGL3-DGK control and Bt 2 cAMP-treated group, respectively. B: Depiction of Ϫ 1,000 to Ϫ 700 bp of the DGK promoter. Putative SF1/SREBP binding sites are denoted by ovals and labeled M1 to M6. C: H295R cells were transiently transfected with wild-type or mutant (M1 to M6) pGL3-DGK , pCMV6-GFP-SF-1, and pRL-CMV and luciferase activity quantifi ed by luminometry. Data are expressed as the fold change in pGL3-DGK reporter gene activity over the untreated control group mean and represent the mean ± SEM of three separate experiments, each performed in triplicate. Asterisks (*) and carats (^) indicate a statistically signifi cant difference ( P < 0.05) from the untreated control group and untreated SF1-transfected group, respectively. D: Luciferase activity was quantifi ed in lysates that were isolated from H295R cells that were transfected with wild-type or mutant (M1, M2, and M3) pGL3-DGK , pRL-CMV, and pcDNA3.1-SREBP1c expression plasmids. Changes in DGK promoter activity are normalized to Renilla luciferase activity and graphed as fold change over wild-type untreated control group. Asterisks (*) and hash (#) indicate a statistically signifi cant difference ( P < 0.05) from the untreated control group and the untreated SREBP1c-transfected group, respectively. for 24-72 h with Bt 2 cAMP. As shown in Fig. 6B , the total PA concentration was increased by 3-fold at the 72 h time point, with a concomitant 48% decrease in the cellular amount of DAG ( Fig. 6C ). Consistent with the increase in total cellular PA, nuclear PA concentrations were also increased with Bt 2 cAMP treatment in a time-dependent manner . Finally, to assess the relative contribution of DGK to the cAMP-stimulated increase in PA, we quantifi ed the concentrations of PA in wild-type and DGK knockdown ( Fig. 6B ) H295R cell lines. Knockdown of DGK mutation of regions M1 (997/ Ϫ 986) and M6 (760/ Ϫ 751) increased basal luciferase activity ( Fig. 3C ). Compared with the wild-type promoter, mutation of M2 ( Ϫ 976/ Ϫ 967), M3 ( Ϫ 910/ Ϫ 904), and M4 ( Ϫ 817/ Ϫ 809) signifi cantly attenuated the SF1 response. As shown in Fig. 3D , mutation of regions M1 and M2 signifi cantly reduced SREBP1c-stimulated DGK reporter gene activity. In contrast to the requirement of region M3 for SF1-dependent transcription, mutation of M3 had no effect on SREBP1-stimulated transcriptional activity of the DGK reporter gene. Further, mutation of M4, M5, and M6 were unable to reduce SREBP1-stimulated DGK luciferase activity. Collectively, these studies indicate that M2 ( Ϫ 976/ Ϫ 967), M3 ( Ϫ 910/ Ϫ 904), M4 ( Ϫ 817/ Ϫ 809) contribute to SF1-dependent transactivation, whereas SREBP1 requires region M2 .

cAMP promotes the recruitment of SF1 and SREBP1 to the DGK promoter
We next determined the effect of cAMP stimulation on the recruitment of SF1 and SREBP1 to the endogenous DGK promoter by performing ChIP assays using chromatin isolated from H295R cells that were treated with 0.4 mM Bt 2 cAMP for 1 h and found that Bt 2 cAMP increased the acetylation of histone H4 ( Fig. 4A ). cAMP stimulation promoted the enrichment of SF1 and SREBP1 at the DGK promoter by 3.8-and 3-fold, respectively. The proximity of the SF1 and SREBP1 binding sites on the DGK promoter ( Fig. 3B ) and the effect of mutating the M2 region on the ability of both SF1 and SREBP1 to increase DGK reporter gene activity ( Fig. 3C, D ) promoted us to determine if the two proteins interact. As shown in Fig. 4B , FLAG-tagged SREBP1c coimmunoprecipitates with GFP-tagged SF1.

Silencing SF1 or SREBP1 represses DGK mRNA
Next we stably knocked down the expression of SF1 in the H295R cell line and determined the effect on DGK gene expression. Suppressing SF1 abolished both basal and Bt 2 cAMP-stimulated DGK protein ( Fig. 5A ) and mRNA ( Fig. 5B ) expression. Interestingly, silencing SF1 also attenuated the expression of SREBP1 ( Fig. 5A, B ), suggesting that the receptor regulates the expression of SREBP1 in the human adrenal cortex. Consistent with the effect of suppressing SF1 on DGK expression, silencing SREBP1 attenuated DGK protein ( Fig. 5C ) and mRNA ( Fig. 5D ), demonstrating that SREBP1 is required for both basal and cAMP-stimulated DGK transcription. Finally, silencing SREBP1 had no effect on the expression of SF1 ( Fig. 5C ).

cAMP increases DGK nuclear expression and PA concentration
In agreement with our previous fi ndings ( 25 ), and as shown in Fig. 6A , DGK is located in both the cytoplasmic and nuclear compartments of H295R cells. However, although Bt 2 cAMP increased the amount of DGK in the cytoplasm by 1.2-fold, there was a 1.8-fold increase in nuclear DGK protein expression ( Fig. 6A ). To determine if the effect of cAMP on DGK expression led to an increase in activity, we quantifi ed PA concentrations in cells treated Fig. 4. cAMP stimulates the recruitment of SF1 and SREBP1 to the DGK promoter. A: H295R cells were incubated with 0.4 mM Bt 2 cAMP, cross-linked with formaldehyde, and the sheared chromatin immunoprecipitated with antibodies against anti-SF1, antiacetyl histone H4, or anti-SREBP1 and recruitment to the DGK promoter ( Ϫ 1,000/ Ϫ 700) assessed by qPCR and normalized to the ⌬ Ct values of input DNA. Data are expressed as fold change over untreated control and represent the mean ± SD of four separate experiments, each performed in duplicate. A representative gel of PCR reaction is shown where reactions were subjected to agarose (2%) gel electrophoresis and the EtBr-stained PCR products (top bands are output and lower bands input) imaged using a VersaDoc scanner (Bio-Rad). B: CV1 cells were transfected with expression plasmids for GFP-tagged SF1 and FLAG-tagged SREBP1a or SREBP1c and harvested 48 h after transfection. Lysates were subjected to IP using an anti-FLAG antibody and protein A/G agarose. Immobilized proteins were washed, separated by SDS-PAGE, and analyzed by Western blotting. Blots were hybridized to anti-SF1 (upper and lower panel) or anti-FLAG (5% of input lysates; middle panel) antibodies. Shown are representative blots of coimmunoprecipitation experiments that were performed on fi ve separate occasions, each time in duplicate.
blotting for DGK , SF1, SREBP1, and GAPDH. D: RNA isolated from wild-type and SREBP1 knockdown H295R cells was subjected to qRT-CPR analysis. Data are graphed as fold change in DGK , SREBP1, or SF1 expression mRNA expression normalized to the mRNA expression of ␤ -actin and represent the mean ± SEM of three separate experiments, each performed in triplicate. *Statistically different from untreated control group, P < 0.05. reduced both basal and cAMP-stimulated cellular PA concentrations ( Fig. 6B ) and resulted in prevention of the cAMP-stimulated reduction in DAG ( Fig. 6C ). Taken together, our data suggested that DGK plays a major role in the cAMP-dependent increase in PA production.

DISCUSSION
DGKs modulate the concentration of DAG and PA, key second messengers in numerous signaling pathways (48)(49)(50)(51)(52). Recent studies have revealed that DGKs regulate immunity, infl ammation, and the nervous system (53)(54)(55)(56)(57), and aberrant DGK activity is implicated in the etiology of type 2 diabetes, cardiovascular disease, and cancer ( 49,58,59 ). We have previously identifi ed a role for DGK in glucocorticoid production. By virtue of its ability to produce PA, a ligand for the nuclear receptor SF1, DGK regulates the transcription of multiple genes required for cortisol biosynthesis, including CYP17A1 ( 25 ). Our present studies provide a further support that phospholipid metabolism plays a key role in cAMP-dependent steroidogenesis. We demonstrate that the expression of DGK is induced by cAMP ( Fig. 1A ). Although DGK ␣ , DGK ␥ , DGK ␦ , DGK , DGK , DGK , and DGK are expressed in H295R cells ( 25 ), the mRNA expression of these isoforms is not affected by Bt 2 cAMP. In agreement with our previous fi ndings ( 25 ), our data suggest that DGK is the main PA source in cAMP-stimulated human adrenocortical cells ( Fig. 6B ). We previously demonstrated that cAMP rapidly induced nuclear DGK catalytic activity within 5 min ( 25 ). Herein, we showed that cAMP, in addition to an acute effect on DGK enzymatic activity and activation of the cAMP signaling pathway, also chronically increased the expression ( Fig. 1 ) and activity ( Fig. 6A ) of DGK .
Luciferase reporter assays revealed that SF1 and SREBP1c increased the transcriptional activity of a DGK reporter gene ( Fig. 3 ). The activation of DGK luciferase activity and the recruitment of the receptor to the endogenous DGK promoter ( Fig. 4A ) suggest that cAMP signaling may activate a feed-forward mechanism that enables the sustained activation of SF1 target genes that are required for glucocorticoid production. We envision that optimal steroid hormone production requires not only a rapid increase in nuclear PA production in response to ACTH/ cAMP ( 25 ), but also a mechanism to facilitate the continued ability of SF1 to activate target gene transcription. One mechanism to achieve SF1 activation is to allow for an increase in DGK expression, and subsequently PA production . SF1 plays an essential role in inducing the transcription Fig. 5. Silencing SF1 and SREBP1 suppresses DGK gene expression. A: H295R wild-type and SF1 knockdown (k.d.) cells were treated with 0.4 mM Bt 2 cAMP for 48 h and cell lysates were harvested and analyzed by SDS-PAGE (8%), followed by Western blotting for DGK , SF1, SREBP1, and GAPDH. B: Real time RT-PCR was used to assess the mRNA expression of DGK , SREBP1, and SF1 using total RNA that was isolated from wild-type and SF1 knockdown H295R. Data are graphed as fold change in DGK , SREBP1, or SF1 expression mRNA expression normalized to the mRNA expression of ␤ -actin and represent the mean ± SEM of three separate experiments, each performed in triplicate. *Statistically different from untreated control group, P < 0.05. C: Wild-type and SREBP1 knockdown cells were treated with 0.4 mM Bt 2 cAMP for 48 h and cell lysates were harvested and analyzed by SDS-PAGE and Western of multiple steroidogenic genes, including cytochrome CYP17A1 in the adrenal cortex and gonads. The ability of SF1 to activate target genes is regulated by mechanisms including coregulatory proteins (60)(61)(62)(63), posttranslational modifi cation ( 27,28,(64)(65)(66)(67)(68), and ligand binding ( 25,(69)(70)(71)(72).
SREBPs are considered as master regulators of lipid metabolism. In general, SREBP target genes include cholesterol biosynthetic (e.g., HMG-CoA synthase, LDLR receptor) and lipogenic genes (e.g., acetyl-CoA carboxylase, fatty acid synthase). However, we have also previously shown that SREBP1 is recruited to the CYP17A1 promoter in response to stimulation by sphingosine-1-phosphate ( 47 ). Our current studies demonstrate that SREBP1 is recruited to the DGK promoter ( Fig. 4A ) and is required for both basal and cAMP-stimulated DGK expression ( Fig. 5C, D ).
Interestingly, we also found that SREBP1 and SF1 interact ( Fig. 4B ), suggesting coordinated action between these two transcription factors. The ability of SF1 to act cooperatively with other transcription factors is well documented. SF1 synergizes with several transcription factors, including GATA transcription factors (73)(74)(75)(76), cAMP regulatory element binding proteins (77)(78)(79), AP1 family members ( 80 ), and ␤ -catenin ( 81 ). Signifi cantly, the likelihood of a physical interaction between SF1 and SREBP1 is supported by studies demonstrating that both SREBP1 and SREBP2 interact with hepatic nuclear factor 4 ( 82 ) and with the liver receptor homolog (LRH)1 ( 83 ). LRH1 and SF1 belong to the NR5A subfamily of nuclear receptors and share greater than 90% conservation in the DNA binding domain and are >50% conserved in the ligand binding domain ( 71,84 ), so it is not surprising that SF1 also interacts with SREBP1. However, despite similarities in the ability of the two NR5A family members to interact with SREBP1, the functional consequences on target gene expression differ. Whereas we show herein that SREBP1 and SF1 cooperate in the activation of DGK reporter gene activity ( Fig. 3A ), SREBPs inhibit the ability of LRH1 to activate target genes in HepG2 and Huh7 hepatoma cells by preventing the interaction of LRH1 with the coactivator PGC1 ␣ (peroxisome proliferator-activated receptor ␥ coactivator 1 ␣ ) ( 83 ). Fig. 6. cAMP increases PA production. A: H295R cells were grown on 10 cm dishes and treated with 0.4 mM Bt 2 cAMP and the cytoplasmic and nuclear fractions isolated for SDS-PAGE and Western blotting for DGK , ␤ -tubulin, and lamin. Data graphed represent densitometric analysis of DGK cytoplasmic and nuclear expression, normalized to ␤ -tubulin and lamin expression, respectively. B: Wild-type or DGK knockdown (kd) cells were treated with Bt 2 -cAMP for 24-72 h and the cellular or nuclear lipids isolated for quantifi cation of PA. The cellular or nuclear amount of PA was normalized to the protein concentration. Data graphed represent the mean ± SEM of three separate experiments, each performed in triplicate. *Statistically different from untreated control group, P < 0.05. Inset: Representative Western blot of controls and tetracycline treated H295R cells demonstrating decreased DGK protein levels. C: Wild-type or DGK knockdown H295R cells were grown on 6-well plates and treated with 0.4 mM Bt 2 cAMP for 72 h and the cellular content of DAG quantifi ed by ELISA. The graphed data represent the mean ± SEM of three independent experiments, each performed in triplicate. of adrenocortical cells to produce PA in response to cAMP signaling. However, these fi ndings also suggest that other DGK isoforms or members of the phospholipase D family may also contribute to the increased biosynthesis of PA. In an elegant study recently reported by Mitra et al. ( 96 ), targeted disruption of the PA phosphatase lipin1 in adipocytes revealed a novel role for the transcriptional coactivator and lipid phosphatase in the regulation of cAMP/PKA signaling. These fi ndings provide support for the role of lipid-metabolizing enzymes as key regulators, not only of lipid homeostasis, but also of signal transduction and cellular processes.
In summary, we found that the expression of DGK is induced by cAMP. Both SF1 and SREBP1 are required for constitutive and cAMP-stimulated DGK expression. Additionally, SF1 is a novel regulator of SREBP1 expression. Given the role of DGK in synthesizing the agonist for SF1 ( 25 ), our studies identify a feed-forward mechanism by which the capacity of adrenocortical cells to produce PA in response to cAMP is regulated by SF1.
We also observed that silencing SF1 in the H295R cell line suppresses the expression of SREBP1 ( Fig. 5C, D ). Microarray analysis (K. Cai et al., unpublished observations) revealed that silencing SF1 reduced the expression of several genes in the SREBP regulatory pathway, including SREBP2, insulin induced gene 1 (INSIG1), and SREBP cleavage-activating protein, suggesting a role for the nuclear receptor in regulating cholesterol homeostasis in the adrenal cortex. These fi ndings are inconsistent with studies performed in Huh7 human hepatoma cells demonstrating that silencing LRH1 led to an increase in the expression of SREBP target genes when the cells were cultured in cholesterol-free media ( 83 ). However, further studies are required to delineate the role of SF1 in regulating the expression of SREBP1.
Our data demonstrate that the cellular content of PA increases in response to Bt 2 cAMP treatment, concomitant with a decrease in DAG ( Fig. 6 ). The time course of this increase supports a role for cAMP-stimulated DGK transcription in mediating PA production. However, given our previous studies demonstrating that Bt 2 cAMP rapidly increases nuclear PA ( 25 ), it is likely that activation of the cAMP signaling pathway acutely regulates DGK activity and chronically regulates DGK expression . Indeed, we have preliminary mass spectrometric evidence that DGK is phosphorylated at multiple sites (D. Li et al., unpublished observations). Published fi ndings from other laboratories have demonstrated that phosphorylation plays a key role in regulating DGK activity, thus it is plausible that posttranslational modifi cation also modulates DGK function. PKA and PKC have been shown to phosphorylate DGK in COS7 cells ( 85 ). Phosphorylation of DGK ␣ by the tyrosine kinase Src confers hepatocyte growth factor-induced cell motility ( 86,87 ), whereas PKC-catalyzed phosphorylation of DGK promotes the dissociation of the lipid kinase from PKC ( 88,89 ). Given that DAG stimulates PKC activity, the association with DGK provides a mechanism to limit the ability of PKC to phosphorylate target proteins. Studies are ongoing to investigate the role of phosphorylation in regulating DGK function in response to activation of the cAMP signaling cascade.
Consistent with our previous studies ( 25 ), and the work of others, DGK is expressed in the nucleus of H295R cells. As shown in Fig. 6B , most of the increase in PA production in response to cAMP at the 24 h time point is due to an increase in nuclear PA biosynthesis, demonstrating the importance of spatially regulated phospholipid metabolism in cell signaling. Because other DGK isoforms also exhibit nuclear localization ( 90,91 ), DGK for example ( 25,(92)(93)(94)(95), it was important to determine the relative contribution of DGK to the increased PA production observed in response to Bt 2 cAMP. Though DGK was the sole isoform whose mRNA expression increased after cAMP stimulation ( Fig. 1A ), it is possible that other isoforms may be positively regulated by cAMP at the posttranscriptional level. H295R cells that were stably expressing a shRNA targeted against DGK exhibited a 50% decrease in basal and Bt 2 cAMP-stimulated concentrations of PA, indicating that DGK plays a prominent role in the capacity