Lipin 1does not require PGC-1α to repress NFATc4, and lipin 1 can suppress both NFATc2 and NFATc4. (A) Myc-tagged PGC-1α (PGC-1) was transfected with FLAG-tagged NFATc4 (NFAT) or FLAG-tagged PPARα (PPAR). Coimmunoprecipitations were performed with anti-myc antibodies and immunoblotted with anti-FLAG (top and bottom panels) or anti-myc antibodies (middle panel). (B) Reporter activity of PPARα or NFATc4 in the presence of PGC-1α and lipin 1 (n = 3 or 2, respectively). *, significant difference (<0.05) from PPARα plus lipin 1 alone. (C) BHK cells were transfected with expression vectors for FLAG-tagged NFATc1 to -c4 and either HA-tagged lipin 1 or the HA-tagged lipin 1 mutant D712E. Coimmunoprecipitations were performed as in Fig. 1A.

Lipin 1 forms a complex with both NFATc4 and PPARα on DNA in vitro and is present at the promoter of NFAT target genes in vivo. (A) BHK cells were transfected with expression vectors for FLAG-tagged NFATc4, HA-tagged lipin 1b, or both. The DNA bound complexes were then analyzed by EMSA. The first lane contains labeled oligonucleotide only, while the second lane is lysates from EGFP-transfected cells. The free probe (free), NFATc4-DNA complex (NFAT), and the lipin 1-NFATc4-DNA complex (Lip-NFAT) and antibody-induced supershift (Supershift) are indicated. The right panel shows the specificity of NFAT binding via competition with 100-fold of excess unlabeled oligonucleotide (lanes C) and verifies the identity of the NFATc4 and lipin 1-NFATc4 bands by preincubation with antibodies specific for the epitope tags of NFATc4 and lipin 1, FLAG (lane F) and HA (lane H), respectively. (B) BHK cells were transfected with expression vectors for FLAG-tagged PPARα and HA-tagged lipin 1b. Gel mobility shift assays performed as described for panel A using an oligonucleotide derived from the PPARα binding site in the apoE III promoter. (C) BHK cells were transfected and binding reactions performed as described above. Where indicated, the DNA-binding reaction was supplemented with recombinant lipin 1 (L) or recombinant lipin 1 that was heat inactivated by boiling for 10 min (Δ). (D) Fully differentiated 3T3-L1 adipocytes were infected with adenovirus expressing LacZ shRNA (LacZ), lipin 1 shRNA (shRNA1), or HA-tagged lipin 1. At 3 to 4 days after infection, adipocytes were harvested for protein analysis. The top panel shows the results of Western blot analysis of lipin 1 expression in LacZ shRNA (LacZ)-, lipin 1 shRNA (shRNA1)-, or HA-tagged lipin 1b-expressing cells. The bottom panel shows results obtained with total Mg²⁺-dependent PAP in adenovirus-infected cells. (E) 3T3-L1 adipocytes infected with adenovirus expressing LacZ shRNA (LacZ), lipin 1 shRNA (shRNA1), or HA-tagged lipin 1 were used to prepare nuclear extracts to analyze NFATc4 binding activity. Where indicated, cold competitor oligonucleotide or antibodies to endogenous NFATc4 were included in the reactions. (F) Semiquantitative ChIP assays for the Tnf, Fabp4, and Gapdh promoters in 3T3-L1 cells infected with adenovirus expressing shRNA targeting LacZ or lipin 1. Either no antibody, antibodies specific for NFATc4, lipin 1, or nonimmune antibodies were used to immunoprecipitate chromatin-bound proteins from untreated, control cells (rows C) or cells treated with A23187 (1 μM) plus PMA (50 nM) for 18 h (A+P). PCR was performed for the indicated number of cycles using primer pairs specific for the Tnf, Fabp4, and Gapdh promoters. Input is PCR amplification of 1% of the amount of lysates used for the immunoprecipitation. The results in the figure are representative of two independent experiments.

Depletion of lipin 1 in 3T3-L1 adipocytes induces the expression of NFAT-responsive genes. (A) Real-time PCR analysis of gene expression in 3T3-L1 cells infected with adenovirus expressing LacZ or one of two different lipin 1 shRNAs (sh1 and sh2). In each experiment the results shown are relative to LacZ expression (n = 3 to 6). (B) Northern blot analysis of the indicated NFAT target genes in 3T3-L1 cells infected with adenovirus expressing shRNA toward LacZ or lipin 1 (shRNA2). At 18 to 24 h before harvest, CsA (5 μg/ml) was added, as indicated, to inhibit NFAT activity. The results for confluent but undifferentiated 3T3-L1 preadipocytes are shown on the left (preadipocyte). (C) Real-time PCR analysis of gene expression in 3T3-L1 cells infected with adenovirus expressing shRNA targeting LacZ or lipin 1 (sh2), and EGFP-tagged VIVIT peptide. Where indicated, the cells were pretreated with PMA (50 nM) plus A23187 (1 μM) for 18 h. The total amount of virus used for infections was kept consistent using LacZ shRNA virus. (D) Real-time PCR analysis of gene expression in 3T3-L1 cells infected with adenovirus expressing HA-tagged lipin 1b. The results were analyzed as described above for panel A, with the results normalized to control LacZ reference on the left (Norm) (n = 3). (E) Real-time PCR analysis of TNF-α expression in 3T3-L1 cells infected with adenovirus expressing HA-tagged lipin 1b, the point mutants I-mut and L-mut, or shRNA2 (sh2). For panels D and E, the Student t test indicated a significant difference (P < 0.05) from LacZ alone (*).

Expression of NFAT target genes in fld mice. RNA was isolated from gonadal fat pads from 42-day-old mice with the indicated genotypes and analyzed by real-time PCR (n = 4 to 5 animals for each genotype).

Lipin 1b interacts with NFATc4 and represses transcriptional activity. (A) BHK cells were transfected with expression vectors for EGFP, FLAG-NFATc4 (NFAT), HA-lipin 1 (lipin), and the lipin 1 deletion mutants lip-ΔC (containing amino acids 1 to 641) and lip-ΔN (containing amino acids 107 to 924). Cell extracts were subjected to immunoprecipitation with either anti-FLAG or anti-HA antibodies. Immune complexes were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to the epitope tag of the indicated proteins. (B) BHK cells were transfected as in panel A, but with FLAG-tagged PPARα (PPAR) instead of NFATc4. Immune complexes were isolated and immunoblotted as in panel A. (C) BHK cells were transfected with the indicated combinations of FLAG-NFATc4, HA-lipin 1, lipin 1 deletion mutants, and the NFAT-tk-luc reporter construct. BHK cells transfected with pSV-βgal served as a control for transfection efficiency. The data shown are the relative light units produced by luciferase activity normalized to β-galactosidase expression and are expressed relative to NFATc4 activity alone. Error bars represent standard error (n = 4). Student t test indicates significant difference (P < 0.05) from NFATc4 alone (*). (D) Reporter activity of PPARα was performed as in C using the ACO3-tk-luc reporter construct (n = 4). The data expressed relative to PPARα alone. (E) BHK cells were transfected with expression vectors for FLAG-NFATc4, HA-lipin 1, and the lipin 1 point mutants D712E, I-mut, and L-mut. Coimmunoprecipitations were performed as in panel A. (F) Transfections and coimmunoprecipitation performed as in panel E, except PPARα was transfected instead of NFATc4. (G) NFATc4 reporter analysis with lipin 1 point mutants was performed as in panel C (n = 3). (H) PPARα reporter analysis with lipin 1 point mutants was performed as in panel D (n = 2).

Lipin 1 represses activated NFATc4 and NFATc4 bound to different composite elements. (A) BHK cells were transfected with the indicated expression vectors for FLAG-NFATc4, constitutively active NFATc4 (FLAG-NFAT-CA), and HA-lipin 1. Coimmunoprecipitations were performed as in Fig. 1A. (B) NFATc4 reporter gene analysis with NFATc4, constitutively active NFATc4, and lipin 1 were performed as described in Fig. 1C (n = 3). (C) Reporter gene analysis of BHK cells transfected with expression vectors for FLAG-NFATc4 and HA-Lipin 1 and a constitutively active calcineurin vector (ΔCnA) as indicated. At 24 h after transfection the indicated cells were treated with 1 μM A23187 for 24 h. NFATc4 reporter analysis was performed as in Fig. 1C (n = 3). (D) Differentiated 3T3-L1 cells, were either untreated (control), or treated with CsA (5 μg/ml), or A23187 (1 μM) plus phorbol myristate acetate (PMA; 50 nM) for 18 h. After treatment, nuclear extracts were prepared and antibodies to endogenous lipin 1 and NFATc4 were used for coimmunoprecipitation. (E) The top panel is a schematic of NFATc binding to two different composite elements. For the bottom panel, BHK cells were transfected with expression vectors for FLAG-NFATc4, HA-C/EBPα, and HA-lipin 1b. Coimmunoprecipitation of NFATc4 was performed as in Fig. 1A. (F) BHK cells were transfected with the indicated expression vectors along with the NFAT:C/EBPα-responsive reporter from the promoter of PPARγ. NFATc4 reporter analysis was performed as in Fig. 1C (n = 4). * and #, significant difference (P < 0.05) from NFATc4 alone (*) or from NFAT plus C/EBPα (#).

TSA reverses lipin 1-mediated repression of NFATc4. (A) BHK cells were transfected with expression vectors for FLAG-NFATc4 and HA-lipin 1b and analyzed for transcriptional activation of NFAT-tk-luc in the presence or absence of 500 nM TSA pretreatment for 24 h (n = 3). (B) BHK cells were transfected as in panel A and subjected to coimmunoprecipitation as in Fig. 1A. (C) BHK cells were transfected with expression vectors for Myc-tagged HDAC1-4 and HA-tagged lipin 1. Reciprocal immunoprecipitations using Myc or HA antibodies were performed, and immune complexes were analyzed as in Fig. 1A. (D) ChIP assays with acetylated histone H3 antibodies were performed on 3T3-L1 adipocytes infected and treated as described in the legend to Fig. 4F.

TNF-α secretion is increased in response to depletion of lipin 1-mediated repression of NFAT. (A) Time course of TNF-α mRNA expression in fully differentiated 3T3-L1 cells infected with adenovirus expressing β-galactosidase (βgal), shRNA construct 1 targeting lipin 1 (shRNA1), and HA-tagged lipin 1 (Lipin 1). At 4 days postinfection, the cells were treated with A23187 (1 μM) for the indicated time. A significant increase (*) or decrease (#) from βgal infected cells (P < 0.05) is indicated (n = 2). (B) Real-time PCR analysis of TNF-α mRNA expression in differentiated 3T3-L1 cells infected with adenovirus expressing shRNA toward LacZ (LacZ) or lipin 1 (shRNA1 and shRNA2) for 4 days. At 18 to 24 h before harvest, the cells were treated with CsA (5 μg/ml), INCA-6 (3 μM), A23187 (1 μM), or A23187 (1 μM) plus CsA (5 μg/ml) (n = 3 to 6). (C) Measurement of TNF-α protein secreted into the media (pg/ml) collected from same cells used for experiments in panel B (n = 4; *, P < 0.001 from LacZ; #, P < 0.001 from LacZ plus A23187).

Age-dependent decline in the expression of lipin 1 in ob/ob white adipose tissue (WAT). (A) Gonadal fat pads from ob/ob and ob/+ mice were harvested for protein analysis at 8, 12, and 19 weeks of age. Shown are Western blots for lipin 1, lipin 2, and actin. (B) Subcutaneous fat pads from ob/ob and ob/+ mice were harvested for protein analysis at 8 and 19 weeks of age. Shown are Western blots for lipin 1, lipin 2, and actin. (C) Total PAP activity in lysates from visceral and subcutaneous WAT of ob/ob and ob/+ mice (n = 3 to 6 for each genotype at each age). A Student t test indicated significant differences between ob/ob and ob/+ mice (*; P < 0.02 for visceral WAT at 8 weeks, P < 0.03 for subcutaneous WAT at 8 weeks, and P < 0.005 for subcutaneous WAT at 19 weeks). The data are expressed as nmol of PA dephosphorylated/min/mg of protein.