Transformer 2β homolog (Drosophila) (TRA2B) Regulates Protein Kinase C δI (PKCδI) Splice Variant Expression during 3T3L1 Preadipocyte Cell Cycle*

Background: PKCδ modulates cellular differentiation and proliferation. Results: Splice factor TRA2B regulates alternative splicing of PKCδI. Conclusion: PKCδI is a gate-keeper of adipocyte differentiation. Significance: Understanding the role and regulation of PKCδI during adipogenesis may contribute toward developing a novel target for managing obesity and its co-morbidities. Obesity is characterized by adipocyte hyperplasia and hypertrophy. We previously showed that PKCδ expression is dysregulated in obesity (Carter, G., Apostolatos, A., Patel, R., Mathur, A., Cooper, D., Murr, M., and Patel, N. A. (2013) ISRN Obes. 2013, 161345). Using 3T3L1 preadipocytes, we studied adipogenesis in vitro and showed that expression of PKCδ splice variants, PKCδI and PKCδII, have different expression patterns during adipogenesis (Patel, R., Apostolatos, A., Carter, G., Ajmo, J., Gali, M., Cooper, D. R., You, M., Bisht, K. S., and Patel, N. A. (2013) J. Biol. Chem. 288, 26834–26846). Here, we evaluated the role of PKCδI splice variant during adipogenesis. Our results indicate that PKCδI expression level is high in preadipocytes and decreasing PKCδI accelerated terminal differentiation. Our results indicate that PKCδI is required for mitotic clonal expansion of preadipocytes. We next evaluated the splice factor regulating the expression of PKCδI during 3T3L1 adipogenesis. Our results show TRA2B increased PKCδI expression. To investigate the molecular mechanism, we cloned a heterologous splicing PKCδ minigene and showed that inclusion of PKCδ exon 9 is increased by TRA2B. Using mutagenesis and a RNA-immunoprecipitation assay, we evaluated the binding of Tra2β on PKCδI exon 9 and show that its association is required for PKCδI splicing. These results provide a better understanding of the role of PKCδI in adipogenesis. Determination of this molecular mechanism of alternative splicing presents a novel therapeutic target in the management of obesity and its co-morbidities.

TRA2B (transformer 2␤ homolog (Drosophila)) is also known as SFRS10 (11). It is highly conserved across human, mouse, and flies. A new nomenclature was introduced by Manley and Krainer (12) for SR proteins in 2010. According to this nomenclature, members of the SR family of splicing factors are numbered according to the chronological order of their discovery. Based on this they have assigned SRSF10 (SR splicing factor 10) name to the splicing factor TASR1/SRp38/SRrp40. This nomenclature is not to be confused with TRA2B/SFRS10 splicing factor discussed here. Mammalian Tra2 has two isoforms: Tra␣ and Tra2␤. Tra2␣ plays a role in sexual differentiation similar to that in Drosophila (13). TRA2B is alternatively spliced to TRA2B1, -2, -3, -4, and -5. TRA2B1 mRNA generates a full-length protein, whereas TRA2B2 through -B5 generate truncated proteins lacking parts of RS (arginine/serine repeats) domain whose function in vivo has not yet been established. The role of TRA2B in alternative splicing is attributed to TRA2B1 (14). TRA2B has two RS domains flanking the central RNA recognition motif domain. TRA2B autoregulates its protein expression (15).
Here, we evaluated adipogenesis and the role of PKC␦I during early phases of differentiation of 3T3L1 preadipocytes. Furthermore, we identified the splice factor mediating the expression of PKC␦I splice variant in 3T3L1 cells.
Flow Cytometry-Cells were trypsinized and washed one time with phosphate-buffered saline (PBS). The cell pellet (containing one million cells) was resuspended in 500 l of PBS and fixed by the slow, dropwise addition of 4.5 ml of ice-cold 70% ethanol while vortexing. Samples were incubated overnight at 4°C to complete fixation and then stored at Ϫ20°C until stained. Fixed cells were centrifuged at 1000 rpm for 5.0 min. The cell pellet was washed twice with PBS. The cell pellet was resuspended in 50 l of RNase A (100 g/ml) and incubated at room temperature for 5.0 min. One ml of PBS was added, and samples were divided to create an unstained negative control for cell cycle analysis. Propidium iodide (50 g/ml in PBS) was added to samples to be stained and samples were incubated at 37°C for 30 min and then analyzed on the Accuri C6 flow cytometer using the FL2 channel. Pulse analysis was used to gate the single cell population and then scatter plot gating was applied to remove debris. Markers were set based on the propidium iodide histogram, and the percentage of G 0 G 1 , S phase, and G 2 /M cells was calculated by the Accuri C6 software.
Western Blot Analysis-Protein lysates were obtained from 3T3L1 cells using lysis buffer containing protease inhibitors.
PCR-Total RNA was isolated from 3T3L1 cells with RNA-Bee (Tel Test Inc.) as recommended by the manufacturer. Two g of RNA was used to synthesize first-strand cDNA with oligo(dT) primer or random hexamer primer and the Omniscript R kit (Qiagen). The following primers were used in PCR for the splicing minigene: PKC␦ forward primer 5Ј-CATCCTAGGT-CCTGCGACAA-3Ј; ␤-actin forward primer 5Ј-CTTCATTG-ACCTCAACTCATG-3Ј; reverse primer 5Ј-TGTCATGGAT-GACCTTGGCCAG-3Ј; SD primer 5Ј-TCTGAGTCACCTG-GACAACC-3Ј; SA primer 5Ј-ATCTCAGTGGTATTTGTG-AGC-3Ј. After PCR, 5% of products were resolved on 6% PAGE gels and detected by silver staining. The PCR reaction was optimized for linear range amplification to allow for quantification of products. Data were analyzed using AlphaView software.
Quantitative Real-time qPCR-Total RNA was isolated from 3T3L1 cells using RNAzol according to the manufacturer's protocol (TelTest Inc.). Two g of RNA were reverse-transcribed with an Omniscript R kit (Qiagen) using oligo(dT) primers or random hexamer primers (for the RNA-immunoprecipitation (RIP) assay). QPCR was performed using 1.0 l of cDNA and Maxima SYBR Green/Rox qPCR master mix (Thermo Scientific). The primers used were: PKC␦I sense primer 5Ј-ACATC-CTAGACAACAACGGGAC-3Ј and antisense 5Ј ACCACGT-CCTTCTTCAGACAC-3Ј; PKC␦II sense primer 5Ј-CACCAT-CTTCCAGAAAGAACG-3Ј and antisense 5ЈTCGCAGGTCT-CACTACTGCCTTTTCC-3Ј; GAPDH sense primer 5Ј-TGA-CGTGCCGCCTGGAGAAAC-3Ј and antisense 5Ј-CCGGCA-TCGAAGGTGGAAGAG-3Ј. Amplification was performed on the ViiaA 7 (Applied Biosystems). Real-time PCR was then performed in triplicate on samples and standards. The plate setup included a standard series, no template control, no RNA control, no reverse transcriptase control, and no amplification control. After primer concentrations were optimized to give the desired standard curve and a single melt curve, relative quotient was determined using the ⌬⌬C T method with GAPDH as the endogenous control and day 0 as the calibrator sample. Experiments were repeated four times.
siRNA Transfection-PRKCD siRNA (ID: 103702), PKC␦IIspecific siRNA (ID: 444054), and scrambled siRNA were purchased from Ambion. TRA2B siRNA (ID: SR408282 A, B, C) and its scrambled control were purchased from Origene. In our experiments SR408282 A gave optimal results. These siRNA were previously validated for specificity, and off-target gene effects were eliminated. The siRNAs were transfected for 48 -72 h using siPORT NeoFX transfection agent or siTRAN transfection agent.
Transient Transfection of Plasmid DNA-3T3L1 preadipocytes were trypsinized, and cell pellets were collected in 100 l of Nucleofector solution (Lonza) and combined with plasmid DNA (2 g). The cell/DNA solution was transferred to a cuvette, and the program was initiated (0.34kV, 960 microfarads). Medium (500 l) was added immediately, and cells were gently transferred to 60-mm plates and allowed to differentiate. In some experiments Trans-IT 3T3™ (Mirus Bio LLC) transfection agent was used according to the manufacturer's instructions.
Oil Red O Staining-3T3L1 preadipocytes were washed with PBS and fixed with 10% formalin for 30 min. The cells were then rinsed and incubated with Oil Red O staining solution (Adipogenesis Assay kit, Millipore) and incubated for 15 min. After washing, images were captured with Nikon confocal microscope. For quantification, 250 l of dye extraction solution was added and incubated for 30 min. Absorbance was read at 520 nm.
Construction of pSPL3-PKC␦ Minigene-The pSPL3 vector was modified to remove cryptic 5Ј splice sites as described in our previous publication (10). The pSPL3 vector was digested with BamHI (in the MCS) and NheI. Primers to amplify genomic PKC␦ from 3T3L1 cells flanked mouse PKC␦ exon 9 (101 bp) and were designed to include the BclI site in the forward primer (in bold type below) and BcuI site in the reverse primers (in bold type). The forward primer was designed to amplify 49 bp of 3Ј intronic sequence such that the product contained the branch point and 3Ј splice site and the reverse primer included 123 bp of 5Ј intronic sequence (splicing minigene A) or 34 bp of 5Ј intronic sequence (minigene B). The primers were: forward primer 5Ј-TGGTGATCAAGGAATG-AGACCTGGGAGACC-3Ј; reverse primer Minigene A 5Ј-AGA-ACTAGTTTTCAGTCTACATGACTCCC-3Ј; reverse primer Minigene B 5Ј-GATACTAGTAAAGAGATATGTGACCC-AGC-3Ј. The products were verified by sequencing and ligated into the digested pSPL3 vector. The overhangs of the selected restriction enzymes hybridized, and this enabled cloning of the PCR product in the proper orientation. The resulting splicing minigenes were verified by restriction digestion and sequencing.
Mutation of Minigene-Mutation was created at the TRA2B site on the splicing minigene A using the QuikChange II sitedirected mutagenesis kit (Agilent Technologies # 200523-5) according to the manufacturer's instructions. The primers used to create the mutation were 5Ј-GGAATATACCAGGGATTT-GAGCCTAAGCCAGAAGTCTCTGGGAGT-3Ј (sense) and 5Ј-ACTCCCAGAGACTTCTGGCTTAGGCTCAAATCCC-TGGTATATTCC-3Ј (antisense). Mutation to the plasmid was confirmed by DNA sequencing. The mutated minigene is referred to as pSPL3-PKC␦**T2b minigene.
RIP Assay-The RIP kit was purchased from Sigma, and the protocol was followed as per the manufacturer's instruction. Tra2␤ antibody was purchased from Sigma, SNRNP70 antibody was from Millipore, and IgG antibody was included in kit (Sigma). Cell lysate (10%) was removed for the input sample. Immunoprecipitation was performed with 2 g of Tra2␤ antibody, SNRNP70 antibody (positive control), or IgG antibody (as the negative control). RNA was purified and treated with DNase to remove genomic DNA. SYBR Green real-time qPCR was performed as described above using PKC␦ primer sets and primers for U1 RNA, the binding partner for the positive control SNRNP70. The primer sequences for U1 RNA are: forward 5Ј-TCCCAGGGCGAGGCTTATCCATT-3Ј and reverse 5Ј-GAACGCAGTCCCCCACTACCACAAAT-3Ј. The yield (% input) and specificity (-fold enrichment) was calculated using Excel™ template for RIP from Sigma.
Statistical Analysis-The PCR gels for the splicing minigene assay and the Western blots were analyzed using AlphaView software from ProteinSimple™. Experiments were repeated three to five times for reproducibility. PRISM™ software was used for statistical analysis. Two-way analysis of variance or matched Student's t test was used in the analysis. A level of p Ͻ 0.05 was considered statistically significant.

RESULTS
Expression Pattern of PKC␦I during in Vitro Differentiation of 3T3L1 Preadipocytes-We previously showed that expression levels of PKC␦ splice variants PKC␦I and PKC␦II switched by day 4 of differentiation (2). Here we sought to evaluate the role of PKC␦I during adipogenesis. Using primers specific to PKC␦I, our real time qPCR results ( Fig. 1) indicate that PKC␦I levels were high in preadipocytes and started declining after day 4 when the cells underwent terminal differentiation.
PKC␦I Regulates Cell Cycle in 3T3L1 Cells-Our results indicated that PKC␦I levels were higher during 3T3L1 preadipocyte mitotic clonal expansion (0 -48 h upon hormonal induction). To determine its role in cell cycle, we knocked down the expression using PRKCD siRNA (25 nM; transfected on day Ϫ1 or scrambled siRNA (25 nM), and cells were harvested 0 -24 h of differentiation). Using SYBR Green real time qPCR, we showed PRKCD siRNA decreased PKC␦I levels, whereas PKC␦II levels remained unaffected. Using flow cytometry, we determined the percentage of cells in the G 0 /G 1 , S, and G 2 /M phase in 3T3L1 control (untreated), scrambled siRNA control, and PRKCD siRNA-transfected cells. Our results indicated that PRKCD siRNA decreased the percentage of cells in the G 2 M phase (Fig.  2). The cells transfected with scrambled siRNA did not differ significantly from the control (untreated) cells indicating that FIGURE 1. 3T3L1 preadipocytes were differentiated in vitro and RNA was isolated every 2 days from day 0 to day 10. Real-time qPCR was performed in triplicate using PKC␦I-specific primers and GAPDH as the internal control. The graph shows relative quantification (RQ) for PKC␦I over 0 -10 days. The experiment was independently repeated five times with similar results. Statistical analysis performed by two-way analysis of variance; ***, p Ͻ 0.0001 highly significant within the group.
transfection of siRNA did not affect differentiation. This indicated an exit of the cell cycle with knockdown of PKC␦I.
PKC␦I Is an Inhibitor of Adipocyte Differentiation-Because our results indicated a role of PKC␦I in preadipocyte differentiation, we transfected PRKCD siRNA along with its scrambled control as described above. Cells were differentiated to day 3. Formation of lipid droplets was evaluated with Oil Red O stain-ing. Images were taken using a Nikon microscope, and the amount of Oil Red O staining was also quantified (see "Experimental Procedures"). Our results (Fig. 3, a and b) indicated that knockdown of PRKCD increased Oil Red O staining compared with control cells, indicating an increase in differentiation in the PRKCD siRNA cells. The cells transfected with scrambled siRNA did not differ significantly from the control (untreated) FIGURE 2. PRKCD siRNA or scrambled siRNA was transfected on day ؊1 in 3T3L1 cells and maintained in culture with differentiation media. Cells were harvested at 0, 6, 12, or 24 h and analyzed. a, SYBR Green real time qPCR using primers specific for PKC␦I or PKC␦II. qPCR results indicate decreased PKC␦I at significant levels at 24 h (day 1 of differentiation). PKC␦II expression was very low in this time period and was not affected by PRKCD siRNA. RQ, relative quantification. b, cell cycle analysis at 0, 6, 12, and 24 h of initiation of differentiation. Cell cycle analysis was performed with propidium iodide staining in flow cytometry, and the graph represents the percentage of cells in either G 0 G 1 , S, or G 2 M phase at the indicated times. Experiments were repeated four times with similar results. Statistical analysis was performed by two-way analysis of variance; ***, p Ͻ 0.0001 highly significant between 24 h scrambled (Scr) siRNA control (Con) and PRKCD siRNA. c, Western blot (IB) analysis of the above samples at 24 h using anti-PKC␦ antibody or ␤-actin. PRKCD siRNA decreased PKC␦I; PKC␦II (expected at 80 kDa) was not detected at 24 h. The experiment was repeated three times with similar results. NOVEMBER  cells. To validate that a decrease in PKC␦I levels promoted adipocyte differentiation, we analyzed PPAR␥ and adiponectin, the markers for adipocyte differentiation, in cells transfected with PRKCD siRNA as described above. Using Western blot analysis (Fig. 3c), our results indicated an increase in PPAR␥1 and -2 and adiponectin along with a decrease in PKC␦I levels. PKC␦II levels were not affected by PRKCD siRNA. Although PKC␦II is not significantly expressed during early differentiation process (0 to 72 h), as a control we transfected either PKC␦II siRNA or PRKCD siRNA on day 0, and cells were analyzed by SYBR Green real time qPCR on day 3 as shown (Fig.  3d). Results showed no significant changes in PPAR␥1/2 with PKC␦II siRNA, whereas PRKCD siRNA results were in agreement with the protein levels. These results suggest that inhibition of PKC␦I allowed the cells to exit the cell cycle and initiate terminal differentiation in 3T3L1 cells.

PKC␦I Regulated by TRA2B Affects Cell Cycle in 3T3L1 Adipocytes
TRA2B Splice Factor Expression in 3T3L1 Cells-Our previous study indicated that the expression patterns of the two alternatively spliced variants of PKC␦ (PKC␦I (via utilization of 5Ј splice site I on exon 9) and PKC␦II (via utilization of 5Ј splice site II on exon 9)) are distinct in 3T3L1 cells. Alternative splicing is regulated by the interaction of trans-factors with the ciselements on the pre-mRNA. To identify the splice factor involved in PKC␦I splicing during early differentiation stage in preadipocytes, we used 16H3 antibody (Millipore). This widely used antibody by RNA biologists detects multiple SR proteins ranging from 20 to 75 kDa. Our results showed a protein at ϳ35 kDa whose pattern corresponded to the expression of PKC␦I, i.e. high expression on day 0 and then tapering down during the following days. Using specific antibodies, our results indicated that SFRS10 (also known as TRA2B/Tra2␤) expression was high on day 0 followed by a gradual decline (Fig. 4a). SRSF1 (also known as SF2/ASF) and SRSF2 (also known as SC35), whose molecular mass, is also around ϳ35 kDa were also individually analyzed for their expression patterns in 3T3L1 cells, and data showed that the PKC␦I levels expression did not concur with their expression patterns (data not shown). These results indicated that Tra2␤ expression was concurrent with PKC␦I expression in 3T3L1 cells. Using computational analysis, we determined that the consensus binding sequence of Tra2␤ (17) was present on PKC␦I exon 9 (Fig. 4b).
Tra2␤ Mediates PKC␦I Splicing-Because our results above indicated a potential role of TRA2B in PKC␦I splicing, we sought to evaluate this further. We transfected TRA2B siRNA (Origene; 10 nM) on day Ϫ1 along with its scrambled control, and harvested the cells on day 2 of 3T3L1 in vitro differentiation. Our results (Fig. 5a) show a decrease of PKC␦I levels with TRA2B siRNA, whereas PKC␦II levels remain unchanged. We confirmed the results with SYBR Green qPCR (Fig. 5b). Next, we overexpressed TRA2B (2 g) on day 6 when both PKC␦I and TRA2B levels are lower in differentiating 3T3L1 cells and analyzed cells on day 8. Our results (Fig. 5c) indicated an increase in PKC␦I levels with overexpression of TRA2B. These results demonstrate that TRA2B mediated PKC␦I splicing.
Inclusion of PKC␦I Exon in a Splicing Minigene-Splicing minigenes are advantageous to study alternative splicing events without the influence of endogenous factors. Hence to evaluate the role of TRA2B in PKC␦I alternative splicing, we cloned heterologous splicing minigenes which contained PKC␦I exon 9 and its flanking 3Ј and 5Ј sequences cloned into a splicing vector pSPL3 (see "Experimental Procedures"). The splicing minigene A contained the alternative 5Ј splice site II, whereas splicing minigene B was trimmed to contain minimum 5Ј flanking sequences without additional 5Ј splice sites. Splicing minigenes A or B or pSPL3 vector was transiently transfected on day 0. 3T3L1 cells were differentiated for 48 h. RNA was isolated and RT-PCR performed using primers for SD (splice donor exon of pSPL3) and SA (splice acceptor exon of pSPL3). Our results show (Fig. 6) PKC␦I exon inclusion via utilization of 5Ј splice site I in the splicing minigenes A and B in 3T3L1 cells. The 5Ј splice site II was not utilized at this stage of differentiation in 3T3 preadipocytes.
Mutation of PKC␦ Splicing Minigene-Our computational analysis of PKC␦ exon 9 showed the presence of a TRA2B consensus binding sequence gagaagaa. To determine whether Tra2␤ bound to the cis-element to promote PKC␦I splicing, we mutated this TRA2B sequence within the PKC␦ splicing minigene A (referred to as pSPL3-PKC␦**T2B splicing minigene; schematic in Fig. 7a). 3T3L1 cells were transfected on day 0 with either splicing minigene A or mutated pSPL3-PKC␦**T2B splicing minigene along with Tra2␤ (2 g). Cells were then differentiated for 24 h. This enabled quantification of the response of Tra2␤ on PKC␦ exon 9 inclusion as 48 h differentiation in Fig. 6 resulted in maximum PKC␦I inclusion. RNA was collected, and PKC␦I exon inclusion was evaluated by PCR using SD-SA primers. Our results (Fig. 7b) indicated that Tra2␤ increased PKC␦I exon 9 inclusion using 5Ј splice site I in the splicing minigene A and that mutation of TRA2B sequence inhibited inclusion of PKC␦I exon9. This demonstrates that TRA2B mediated splicing of PKC␦I mRNA.
Tra2␤ Binds to PKC␦I Pre-mRNA-To establish that Tra2␤ bound to PKC␦ mRNA, we performed RIP. The RIP assay involves immunoprecipitation of complexes formed endogenously between the RNA-binding proteins along with the associated RNA in vivo. 3T3L1 cells were collected on day 0 when they were 100% confluent. The complex was cross-linked in vivo using formaldehyde. Tra2␤ antibody was used to immunoprecipitate the complex. A negative control (mock IgG IP) or positive control (SNRNP70 IP) for RIP assay was included in the experiments. Supernatant (50 l) after the last wash was ali-FIGURE 3. 3T3L1 preadipocytes were transfected with PRKCD siRNA or scrambled siRNA and maintained in culture through differentiation for 3 days. a, Oil Red O staining for lipid accumulation. b, quantification of absorbance of Oil Red O assay. Statistical analysis was performed by two-tail Student's t test; *** p Ͻ 0.0001 highly significant between day 3 scrambled siRNA control and PRKCD siRNA. c, whole cell lysates were harvested on day 3 and immunoblotted (IB) with anti-PPAR␥, anti-adiponectin, anti-PKC␦ (which can detect PKC␦I and PKC␦II), PKC␦II-specific antibody separately as its expression levels are very low, or anti-␤-actin as indicated. PKC␦I levels were reduced by 80%. Experiments were repeated five times with similar results. The graph represents densitometric units normalized to ␤-actin. d, in separate experiments, PRKCD siRNA, PKC␦II-specific siRNA, or scrambled control was transfected and maintained in culture. Cells were harvested on day 3. SYBR Green real time qPCR was performed for PPAR␥1 and -␥2, adiponectin, PKC␦I, and PKC␦II. RQ, relative quantification. Experiments were repeated five times. Statistical analysis was performed by two-tail Student's t tests; ***, p Ͻ 0.0001 highly significant on day 3 between scrambled siRNA control and PRKCD siRNA for PPAR␥1, PPAR␥2, and adiponectin. quoted for use in a Western blot to confirm IP. The crosslinking was reversed and extracted RNA from the RIP was then used in qPCR. For positive control, qPCR was performed using U1 RNA primers, the binding partner for SNRNP70. Our results (Fig. 8) showed enrichment of PKC␦I with Tra2␤ RIP.

DISCUSSION
PKC␦ has roles in cellular differentiation, proliferation, and apoptosis. The role of PKC␦I splice variant in apoptosis was previously shown by us (9) and others (18,19). Here we evaluated the role of PKC␦I during early stages (0 -48 h) of 3T3L1 differentiation. PKC␦I is the predominant splice variant present in 3T3L1 preadipocytes; PKC␦II is not expressed significantly in the early stages of differentiation. Studies have pointed to the role of PKC␦ in cell cycle. In adipocytes, these were limited to day 0 compared with day 8 (20). To our knowledge no study was undertaken to delineate the roles and mechanism of expression of PKC␦I splice variant during early stages of 3T3L1 differentiation. PKC␦ can be both stimulatory and inhibitory in the cell cycle. PKC effects on cell cycle are generally mediated by downstream signaling cascades such as PI3K/Akt, Erk, or Wnt pathway. PKC␦ affects activity of cyclin-dependent kinases as well as cyclin D1, cyclin E, or cyclin A and promotes phosphorylation of the retinoblastoma (Rb) protein (21,22). It was shown that PKC␦ stimulated G 1 phase cell cycle progression in rat Wistar thyroid cells (23) and that PKC␦ inhibits G 1 cell cycle progression in response to testosterone in coronary smooth muscle cells (24). Phorbol 12-myristate 13-acetate-induced degradation of PKC␦ requires its phosphorylation in NIH3T3 cells (25). PKC␦ is required to maintain G 2 /M checkpoint in keratinocytes in response to UV radiation (26). Here we showed that down-regulation of PKC␦I during 3T3L1 adipogenesis was required for differentiation to adipocytes. The 3T3L1 cells undergo mitotic clonal expansion, which is supported by high levels of PKC␦I and exit from the cell cycle to initiate differentiation is regulated by PKC␦I. Further in-depth FIGURE 4. a, 3T3L1 preadipocytes were differentiated in vitro, and whole cell lysates were collected every 2 days from day 0 to day 10. Western blot (IB) analysis was performed using Tra2␤ antibody, PKC␦ antibody (which can detect PKC␦I and PKC␦II simultaneously), or ␤-actin. The graph shows quantification of normalized densitometric units. The experiment was independently repeated five times with similar results. b, Tra2␤ sequence on PKC␦I exon 9 and schematic of its position (E ϭ exon on the schematic).
analysis would be required to identify the specific cyclin or cyclin-dependent kinases and their phosphorylation cascades as well as cross-talk that mediates this effect of PKC␦I in 3T3L1 cells.
Alternative splicing of PRKCD gene produces the splice variants PKC␦I and PKC␦II in 3T3L1 cells. During adipogenesis, the expression pattern of these PKC␦ splice variants is distinct. PKC␦I is present in preadipocytes, and its expression slowly declines after differentiation, whereas PKC␦II expression increases after terminal differentiation. Alternative splicing is an essential mode for protein diversity and gives the ability to adjust to the constantly changing proteomic needs of the cell in response to development, hormones, and other stimuli. These changes are often a result of the dynamic association of the splice factors with pre-mRNA of the target gene. Pre-mRNA splicing is catalyzed by the spliceosome, which has Ͼ200 components including small nuclear ribonucleoproteins and proteins. Protein factors that function as enhancers and suppressors of splicing are an integral part of the spliceosome (27). In 3T3L1 cells, TRA2B mediates the splicing of PKC␦I mRNA by binding to its cis-element on PKC␦I exon 9. This site is close to the 5Ј splice site, and binding of Tra2␤ promotes the binding of FIGURE 5. a, 3T3L1 preadipocytes were transfected with TRA2B siRNA on day 0 and maintained in culture through differentiation for 2 days. Western blot (IB) analysis was performed with PKC␦ antibody or Tra2␤ antibody. Experiments were repeated four times with similar results. The graph shows quantification of normalized units. b, 3T3L1 preadipocytes were transfected with TRA2B siRNA or scrambled siRNA on day 0 and maintained in culture through differentiation for 2 days. SYBR Green real time qPCR was performed using primers for PKC␦I, PKC␦II, or TRA2B using GAPDH as the internal control. The experiment was independently repeated four times with similar results. Statistical analysis performed by two-tail Student's t test; ***, p Ͻ 0.0001 highly significant between control day2 and TRA2B siRNA day 2 for PKC␦I. RQ, relative quantification. c, Tra2␤ (2 g) was transiently transfected on day 6, and whole cell lysates were collected on day 8. SYBR Green real time qPCR was performed using primers for TRA2B, PKC␦I, or PKC␦II. The experiment was independently repeated four times with similar results. Statistical analysis performed by two-tail Student's t test; ***, p Ͻ 0.0001 highly significant between control day 8 and TRA2B overexpression day 8 for PKC␦I. U1 small nuclear ribonucleoprotein to the 5Ј splice site thereby initiating the splicing reaction. Because several endogenous factors could potentially affect PKC␦I splicing, we also used a heterologous splicing minigene to validate the role of TRA2B in promoting PKC␦I splicing during 3T3L1 adipogenesis. The 5Ј splice site II was not utilized upon TRA2B mutation in the mini-gene, which suggests that utilization of the downstream splice site required splice factors that may be absent during 0 -24 h of differentiation of 3T3L1 preadipocytes. Our studies point to the role of TRA2B in cell cycle progression. It is possible that TRA2B affects splicing of other genes involved in the cell cycle; however, this remains to be elucidated. 1, constitutive splicing of SD to SA; 2, inclusion of PKC␦ exon9 using 5Ј splice site I; 3, product utilizing 5Ј splice site II. The pSPL3 splicing vector or the splicing minigenes were transfected in differentiating adipocytes (day 0), and RNA was harvested on day 2. PCR performed using SD-SA primers. Products were separated by PAGE and were silver-stained for visualization. 5Ј splice site II was not utilized on day 2 in the splicing minigene assay; constitutive splicing of SD to SA serves as a positive control for the assay. Graphs represent the percent exon inclusion calculated as SS I/(SS I ϩ SSII) ϫ 100 in the samples and are representative of five experiments performed separately. FIGURE 7. Mutation of Tra2␤ site on PKC␦ splicing minigene. a, schematic of Tra2␤ binding site mutated within pSPL3-PKC␦ splicing minigene. *** on the schematic indicates mutated Tra2␤ binding site. Arrows indicate primer positions on splice donor exon (SD) and splice acceptor exon (SA). b, either splicing minigene A (Mg A) or pSPL3-PKC␦**T2b splicing minigene was co-transfected along with 2 g of Tra2␤ in differentiating adipocytes (day 0), and RNA was harvested 24 h later on day 1. PCR was performed using SD-SA primers. Products were separated by PAGE and were silver-stained for visualization. Product 1 is SD-SA constitutive splicing; product 2 is PKC␦ exon inclusion using 5Ј splice site I (SSI). Graphs represent percent exon inclusion calculated as SS I/(SS I ϩ SSII) ϫ 100 in the samples and are representative of four experiments performed separately. The experiment was independently repeated five times with similar results.
Regulation of alternative splicing by TRA2B is shown in several systems including LIPN1 in liver (28), splicing of myosin phosphatase targeting subunit 1 in smooth muscle (29), splicing of Tau in neurons (30) and BRCA1 gene (31). TRA2B is also shown to be involved in mouse embryogenesis and spermatogenesis as well as in meiosis in male germ cells (32). Our previous study demonstrated dysregulated PKC␦ alternative splicing in obese adipocytes (1) compared with lean. Furthermore, the adipogenesis program was also dysregulated in obese subjects. Hence, understanding the regulation of expression of PKC␦I by splice factor TRA2B during adipogenesis is important to the study of obesity pathology.