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Molecular Endocrinology, doi:10.1210/me.2003-0391
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Molecular Endocrinology 18 (4): 899-911
Copyright © 2004 by The Endocrine Society

Insulin Regulates Protein Kinase CßII Alternative Splicing in Multiple Target Tissues: Development of a Hormonally Responsive Heterologous Minigene

Niketa A. Patel, Hercules S. Apostolatos, Konrad Mebert, Charles E. Chalfant, James E. Watson, T. S. Pillay, Janet Sparks and Denise R. Cooper

Department of Biochemistry and Molecular Biology (N.A.P., H.S.A., K.M., D.R.C.), College of Medicine, University of South Florida, and The Research Service (J.E.W., D.R.C.), James A. Haley Veterans Hospital, Tampa, Florida 33612; Department of Biochemistry (C.E.C.), Virginia Commonwealth University and Hunter Holmes McGuire Veterans Medical Center, Richmond, Virginia 23298-0614; Institute of Cell Signaling (T.S.P.), University of Nottingham Medical School, Queen’s Medical Center, Nottingham NG7 2UH, United Kingdom; and University of Rochester (J.S.), School of Medicine and Dentistry, Rochester, New York 14642

Address all correspondence and requests for reprints to: Denise R. Cooper, Ph.D. J. A. Haley Veterans Hospital (VAR 151), 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: dcooper{at}hsc.usf.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C ß (PKCß) isozymes and preferentially switches the expression to PKCßII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCßII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCßII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCßII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCßII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AN IMPORTANT SOURCE of protein diversity and control of gene expression is alternative pre-mRNA splicing. Multiple mRNAs can be generated by alternative splicing in response to tissue specificity, growth, or hormonal stimuli. The human genome project predicts 47–60% of the human genes to be alternatively spliced as analyzed by the expressed sequence tags (1, 2). Defective alternative splicing causes a large number of diseases. Hence, the elucidation of the factors and cis-elements regulating alternative splicing is of critical importance.

The spliceosome catalyzes the pre-mRNA splicing reaction within a large multicomponent ribonucleoprotein complex. This complex comprises small nuclear RNAs and associated proteins. The proteins involved in the spliceosome assembly have RNA-binding domains and protein-interaction domains. Splicing of the correct exon is a dynamic process involving the joining of 5'- and 3'-splice sites that define the exon-intron boundary by the interplay of small nuclear riboproteins and other associated proteins with the pre-mRNA sequences (3). Exon recognition can be stimulated or repressed by regulating protein interaction with pre-mRNA sequences called the exonic or intronic splicing enhancers or silencers. Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family can bind to exonic splicing silencers and intronic splicing silencers and function as splicing repressors. The SR (Ser-Arg rich) proteins are non-small nuclear riboproteins, which bind to the exon splicing enhancer and promote the choice of splice sites. Bound SR proteins can then recruit other splicing factors and thereby play an important role in splicing regulation (4, 5, 6).

Protein kinase C (PKC) ßI and -ßII are alternatively spliced products of PKCß pre-mRNA having distinct cellular functions in cell proliferation and differentiation, apoptosis, and transcriptional activation (7). PKC is a serine/threonine kinase comprising 12 different isoforms. Insulin regulates the inclusion of PKCßII exon and promotes the switch of the PKCßII isoform from the -ßI isoform via alternative splicing (8). A STOP codon in PKCßII exon near the splicing junction prevents the translation of the PKCßI exon (9, 10). PKCßII has distinct physiological roles in glucose uptake (11, 12), attenuation of DNA synthesis, and cellular proliferation (13, 14, 15). Further, it has been shown that PKCßII regulates insulin receptor by protein-protein interactions (16). Usually, the inclusion of an alternatively spliced exon is controlled in a tissue-specific, sex-specific, or a developmentally specific manner (17, 18, 19, 20, 21, 22, 23, 24). In this novel scenario, alternative splicing is under hormonal control, i.e. regulated by insulin within 15 min (8). This rapid response is distinct from other hormonally regulated systems that have been described (20, 25, 26).

Our previous work demonstrated that insulin activated additional 5'-splice sites in PKCßII mRNA (27). These transcripts encode the same protein but have varying 5'-untranslated regions. Further, we have shown that insulin regulates endogenous PKCßII exon inclusion via phosphorylation of the splicing factor SRp40 (28). Here we show evidence of a functional heterologous minigene in which insulin regulated the inclusion of PKCßII exon and activated an additional 5'-splice site in distinct cell types such as skeletal muscle, vascular smooth muscle, preadipocytes, embryonic fibroblasts, and hepatoma cells. We demonstrate that this heterologous minigene is regulated by phosphorylation in vivo by a phosphatidylinositol 3-kinase (PI3-kinase) pathway via Akt2 kinase. We have identified the essential intronic sequences required for insulin-regulated PKCßII exon inclusion in the minigene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin-Regulated PKCßII Exon Splicing in Hepatocytes, 3T3-Adipocytes, and Hepatoma Cells
Insulin regulated PKCßII exon inclusion in L6 cells and activated an additional 5'-splice site in a time-dependent manner (27). Alternative splicing of the PKCß gene was further studied in other cell types to evaluate whether insulin regulated endogenous PKCßII expression in other target tissues. Insulin promoted exon inclusion of PKCßII mRNA using splice site I in primary rat hepatocytes and 3T3-L1 adipocytes (Fig. 1Go, B and C). The PI3-kinase inhibitor LY294002 blocked insulin-stimulated exon inclusion in 3T3-L1 adipocytes (Fig. 1CGo). In the human hepatoma HepG2 cells, insulin activated additional upstream PKCßII 5'-splice site (Fig. 1DGo) similar to L6 cells. However, it was difficult to reproduce endogenous exon inclusion in vascular smooth muscle cells (data not shown).



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  		Fig. 1. Insulin-Regulated Endogenous Alternative Splicing of the C Terminus of PKCß pre-mRNA

A, Inclusion of PKCßII exon 17 generates PKCßII mRNA. A STOP codon at the splice junction hinders the translation of the PKCßI exon. Total RNA was isolated from either (B) primary rat hepatocytes, (C) 3T3-L1 adipocytes, or (D) Hepatoma HepG2 cells. RNA (2 µg) was used in the RT-PCR analysis using primers for the last common, C4, domain (exon 16) and antisense primer to the PKCßI exon (indicated by arrows). The PCR product size was 187 bp for PKCßI and 404 bp (splice site I) or 539 bp (splice site II) for PKCßII. Primers for ß-actin were used for normalization. The experiments were repeated two to three times for each cell type and similar results were obtained.

 
Development of an Insulin-Regulated Heterologous Minigene
To dissect the mechanism of regulation of PKCßII alternative splicing by insulin, a PKCßII heterologous minigene was developed to identify insulin-responsive cis-elements in vivo in eukaryotes (Fig. 2AGo). Because the splice variants detected in vivo in L6 and HepG2 cells appeared to use alternative 5'-splice sites, it was postulated that the elements involved in regulation of exon inclusion and splice site selection would reside within the PKCßII exon and its flanking 3'- and 5'-intronic sequences. Hence, the 216-bp PKCßII exon and 1000-bp upstream and 1200-bp downstream flanking intronic sequences were cloned into the pSPL3 vector between the splice site donor and the splice site acceptor exons.



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  		Fig. 2. Insulin-Regulated PKCßII Splicing Minigene

A, A heterologous splicing minigene was constructed by inserting the PKCßII genomic fragment (exon 17) into a multicloning site in between the SD and SA in pSL3. Putative cis-elements were identified by inspection of the sequence and are indicated relative to one another. PT, Pyrimidine tract; AURE, AU-rich Element; ISE, intronic splicing enhancer; CAG/GTG, splice site. B, Two sets of primers were used in the amplification of splice products. Primers SD-SA amplified three products: 263 bp when PKCßII exon was not included; 479 bp (I) when PKCßII exon was spliced using splice site I; 614 bp (II) when PKCßII exon was spliced using splice site II. Primers ßII-SA amplified two products: 352 bp (I) when PKCßII exon was spliced using splice site I; 487 bp (II) when PKCßII exon was spliced using splice site II. C, The pSPL3–32 minigene was transiently transfected into L6 cells. Cells either had no insulin treatment (I0) or were treated with insulin for 30 min (I30). Total RNA was extracted and RT-PCR was performed using primers corresponding to either SD-SA or ßII-SA. D, The PCR products were verified by Southern blot analysis using PKCßII-specific probe as described. Labeled probe hybridized to PCR products as predicted.

 
To determine whether the pSPL3–32 minigene could mimic RNA processing and splice site selection as observed for endogenous PKCßII mRNA, the pSPL3–32 minigene was transiently transfected into rat skeletal muscle (L6) cells. Insulin-responsive inclusion of the PKCßII exon and activation of another upstream splice site was demonstrated in L6 cells using two sets of primers. Primers to splice site donor (SD) and splice site acceptor (SA) amplified three products: 263 bp amplifying SD-SA showing constitutive minigene splicing; 479 bp (I) when PKCßII exon was included using splice site I; and 614 bp (II) when PKCßII exon was included using splice site II. Primers within the PKCßII exon and SA amplified two products: 352 bp (I) when PKCßII exon was included using splice site I; and 487 bp (II) when PKCßII exon was included using splice site II (Fig. 2BGo). When cells were not treated with insulin, only constitutive splicing of the minigene is observed using primers SD-SA whereas no product is detected using primers ßII-SA as the PKCßII exon is skipped (Fig. 2CGo). Both sets of primers detect products resulting from activation of splice sites I and II after insulin treatment. Hence, this minigene contained all the necessary sequences to promote insulin-regulated PKCßII exon inclusion and splice site selection.

Insulin-Regulated PKCßII Minigene in Multiple Target Tissues
Hormonally responsive alternative splicing of endogenous PKCßII mRNA is readily detected in differentiated skeletal muscle cells such as L6 cells (11). However, in rat vascular smooth muscle A10 cells, insulin-induced endogenous splicing of PKCßII isoform was not readily detected. This is presumably due to the complex transcriptional and posttranscriptional regulation of PKCßII mRNA (15). To further test the model in other insulin-responsive cells, the pSPL3–32 minigene was transiently transfected into the rat vascular smooth muscle A10 cells, human hepatoma cell line, HepG2 cells, 3T3-pre-adipocytes, and murine embryonic fibroblasts. Insulin regulated PKCßII exon inclusion and splice site activation of the pSPL3–32 minigene in each cell line (Fig. 3Go). Additionally, splice site II was activated in all cell types by insulin in a time-dependent manner similar to that detected in L6 cells.



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  		Fig. 3. The pSPL3–32 Minigene Was Regulated by Insulin in Various Cell Types

Cells either had no insulin treatment (I0) or were treated with insulin for 15 min (I15), 30 min (I30), or 60 min (I60). RT-PCR was performed using primers for ßII-SA to detect activation of splice sites I and II by insulin. ß-actin levels were equivocal between the treatments. Each experiment was repeated two to four times with similar results to establish activation of splice sites I and II.

 
Interestingly, different cells show distinct responses to insulin treatment. L6 skeletal muscle cells show a preference to utilizing splice site II after 15 min of insulin stimulation whereas in A10 vascular smooth muscle cells, insulin stimulation for 15 min promoted the use of splice site I. Both cell types utilize splice sites I and II with longer insulin treatment. HepG2 hepatomas predominantly use splice site II whereas 3T3-L1 preadipocytes utilize splice site I with longer (30–60 min) insulin treatments. Also, 3T3-L1 preadipocytes and the embryonic fibroblasts show constitutive PKCßII exon inclusion using splice site I without insulin stimulation. Hence five distinct cell types (i.e. skeletal muscle, vascular smooth muscle cells, hepatoma cells, preadipocytes, and embryonic fibroblasts) demonstrate PKCßII exon splice site activation regulated by insulin within a short time of the stimulation, although the use of splice sites I and II are cell and time dependent.

Insulin-Activated PKCßII 5'-Splice Sites
To determine whether the 3'-intron was involved in insulin-regulated 5'-splice site selection, the 3'-flanking intron and a portion of the PKCßII exon including the 3'-splice site were deleted from the pSPL3–32 minigene. A new 3'-splice that the exon can utilize was introduced in the cloning process as described in Materials and Methods. This truncated minigene, pSPL3–17, containing 108 bp of the exon and the intronic sequences flanking the 5'-splice site (Fig. 4AGo) was transfected into L6 and A10 cells. Insulin still regulated the 5'-splice site selection of the PKCßII exon (Fig. 4BGo) in the pSPL3–17 minigene in a time-dependent manner in both the cell types. This suggests that the elements around the 3'-splice site and its flanking intron were not required for activation of the 5'-splice site in vivo by insulin.



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  		Fig. 4. 3' Intronic Sequences Are Not Involved in Insulin-Regulated 5' Splice Site Selection

A, The pSPL3–17 minigene contains 108 bp of the ßII exon and approximately 1200 bp of 5'-intronic sequence. SSI, Splice site I (ATG/GTG); SSII, splice site II (CAG/GTG). B, pSPL3–17 minigene was transiently transfected into L6 skeletal muscle and A10 vascular smooth muscle cells. Cells either had no insulin treatment (I0) or were treated with insulin for 15 min (I15), 30 min (I30), or 60 min (I60). RT-PCR was performed using primers for SD-SA. Insulin activated splice sites I and II in both cell types. The experiment was repeated five times with similar results in both cell types. The graph shows percent maximal ßII-exon inclusion using either splice site I or II, assuming utilization of splice sites I and II as 100% after 60 min of insulin treatment.

 
Insulin Signals via PI3-Kinase to Regulate PKCßII Minigene
Our previous work (28) and studies in 3T3 L1 adipocytes (Fig. 1AGo) indicated that a PI3-kinase-dependent pathway was involved in the in vivo regulation of alternative splicing of PKCßII mRNA by insulin. Because pSPL3–17 contained all the cis-elements required for insulin-stimulated activation of 5'-splice sites, this minigene was transiently transfected into L6 cells. To determine whether the splicing of the minigene was also regulated via a PI3-kinase pathway, PI3-kinase inhibitors, LY294002 (100 nM) or Wortmannin (100 nM), were added 30 min before addition of insulin for 30 min. Moreover, pSPL3–17 was cotransfected with {Delta}p85 {alpha}, the dominant negative mutant of the regulatory subunit of PI3-kinase (30, 31), which lacks the binding site for the p110 catalytic subunit, thereby inhibiting the association of PI3-kinase with insulin receptor substrate-1. Results show inhibition of PKCßII splice site activation, hence demonstrating that splice site selection of the PKCßII minigene was also regulated via the PI3-kinase pathway (Fig. 5Go, A and B).



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  		Fig. 5. 5' Splice Site Selection in pSPL3-17 Minigene Is Regulated by PI3-Kinase Pathway

Insulin (100 nM) was added for 30 min (I30) or cells had no insulin treatment (I0). A, The PI3-kinase inhibitor, LY294002, was added for 30 min before total RNA isolation; or (B) The PI3-kinase inhibitor, wortmannin, was added 30 min before total RNA isolation. The dominant negative subunit of PI3-kinase, p85{alpha} DN, was cotransfected with pSPL3–17. C, Constitutively active Akt2 kinase or (D) Clk/Sty was cotransfected with pSPL3–17. Total RNA was isolated and RT-PCR was performed using primers for ßII-SA in panels A and D. ß-Actin was used as an internal control. RT-PCR was performed using primers for SD-SA in panels B and C. The graph shows percent maximal ßII-exon inclusion using either splice site I or II, assuming utilization of splice sites I and II as 100% after 30 min of insulin treatment. The experiments were repeated at least four times each.

 
Akt2 Is a Downstream Kinase Regulating PKCßII Exon Splice Site Selection
The role of Akt kinase has been implicated in insulin action because it is activated by PI3-kinase (32). Also, SR proteins contain the consensus sequence RXRXXS recognized by Akt kinase. They have been shown to be potential substrates in vitro (33) for Akt kinase. Further, data from our laboratory demonstrate Akt2 kinase phosphorylated SRp40 in vivo (34). To identify the intermediary kinases involved in the phosphorylation cascade, the minigene pSPL3–17 was cotransfected with constitutively active Akt2 kinase (CA-Akt2) into the L6 skeletal cells. CA-Akt2 kinase mimicked the action of insulin (Fig. 5CGo) by promoting exon inclusion and activating the additional 5'-splice site in the minigene.

Clk/Sty Cotransfection Regulated PKCßII Exon Splice Site Selection
The Clk/Sty protein kinase interacts with RNA binding proteins and, in particular, phosphorylates the SR family of splicing factors (35). Mammalian Clk/Sty was shown to phosphorylate SR splicing factors in a physiologically relevant manner. It was suggested that the Clk kinase family could act as a link between signal transduction and regulated splicing (36, 37). Hence, the minigene pSPL3–17 was cotransfected with Clk/Sty in L6 cells. Clk/Sty also activated 5'-splice sites (Fig. 5DGo), suggesting that several kinases could play a role with insulin activating multiple signaling pathways involved in splice site selection.

Binding of SRp40 Is Essential for Insulin-Regulated Splice Site Activation
It was established that the phosphorylation of SRp40 was a key step in the regulation of splice site selection and exon inclusion by insulin (28). However, binding of SRp40 to the pre-mRNA may be exclusive of its phosphorylation and function in PKCßII splicing. To further elucidate the role of SRp40 in splice site selection, its RNA-binding site, TGGGAGCTTGGCTTAGA, in the downstream intron flanking the PKCßII exon [which has a 2-bp mismatch with the sequence predicted by SELEX (38)] was replaced with AGCGAATCATTGAATCC in the pSPL3–32 minigene. This sequence replacement ablated the insulin-stimulated exon inclusion and splice site activation in the minigene (Fig. 6AGo). This emphasizes the requirement of SRp40 binding to its site on the pre-mRNA in addition to its phosphorylation by an upstream kinase in insulin-regulated PKCßII 5'-splice site selection.



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  		Fig. 6. SRp40 Binding and Phosphorylation Affect PKCßII 5' Splice Site Selection

A, The SRp40 binding site in the 5'-intronic sequence was replaced with AGCCGAATCATTGAATCC in the pSPL3–32 minigene. Both the pSPL3–32 and mutated pSPL3–32* minigenes were transiently transfected into L6 cells. Insulin was added for 30 min (I30) or 60 min (I60), or cells were not treated with insulin (I0). RT-PCR was performed using primers for SD-SA. The experiment was repeated five times with similar results. The graphs shows percent maximal ßII-exon inclusion using either splice site I or II, assuming utilization of splice sites I and II as 100% after 60 min of insulin treatment in the pSPL3–32 minigene. The experiments were repeated at least three times with similar results. B, The pSPL3–32 minigene was cotransfected into L6 cells along with increasing amounts (0.8–1.6 µg) of SRp40. Insulin (100 nM) was added 30 min before total RNA isolation. RT-PCR was performed using primers to SD-SA. ß-Actin was used as an internal control. The experiment was repeated four times with similar results. C, The pSPL3–32 minigene was cotransfected into L6 cells along with either SRp40 or constitutively active Akt2 kinase (CA-Akt2). Insulin (100 nM) was added 30 min before total RNA isolation. ß-Actin was used as an internal control. RT-PCR was performed using primers to ßII-SA. The experiment was repeated thrice to ensure reproducible results.

 
Overexpression of SR factors has also been shown to enhance exon inclusion (39). Although we hypothesize that phosphorylation of SRp40 regulates its RNA binding activity, overexpression should mimic, to an extent, insulin effects. Increasing amounts of SRp40 (0.8–1.6 µg/35-mm plate) were cotransfected with pSPL3–32 to observe its influence on splice site selection. Results indicate that although cotransfection of 0.8 µg SRp40 activated splice sites I and II (compared with no insulin treatment), addition of insulin leading to phosphorylation of SRp40 enhanced splice site activation to 100% (Fig. 6BGo). Cotransfection with 1.6 µg SRp40 (per 35-mm plate) activated splice sites I and II (Fig. 6BGo) although amounts higher than 2.4 µg of SRp40 per 35-mm plate suppressed splicing (data not shown).

Further, the constitutively active form of Akt2 kinase, CA-Akt2, was cotransfected along with pSPL3–32 and SRp40 (0.8 µg/35-mm plate) in L6 cells. It mimicked insulin-induced splice site activation (Fig. 6CGo). This provides a link to the downstream kinase cascade leading to the splice site selection in PKCßII mRNA.

Consecutive Deletion of the 5'-Intronic Sequences in PKCßII Minigene Reveals Multiple Regulatory Effects
The contribution of the downstream intronic sequence for activating splice sites I and II was important but unknown. Hence, consecutive deletions were performed on the minigene pSPL3–17 to truncate the 5'-intron and elucidate the importance of other putative elements on the 5'-splice site selection (Fig. 7AGo). GA-rich intronic splicing enhancers, present at the 5'-end of introns, have been implicated in promoting exon inclusion (40). To determine whether these sequences played a role in PKCßII exon inclusion and splice site selection, the minigene pSPL3–18 was truncated after the AU-rich element, thereby eliminating the GA-rich splicing enhancers and then transfected into A10 cells. The pSPL3–18 minigene still demonstrated regulated PKCßII splice site selection (Fig. 7BGo) with activation of splice sites I and II, suggesting no involvement of these putative distal intronic splicing enhancers in insulin action.



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  		Fig. 7. Consecutive Deletions of PKCßII Minigene Reveal Multiple Regulatory Effects

A, Schematic shows the consecutive deletions of putative cis-elements in the 5'-intronic sequence of ßII exon. B, The truncated minigene pSPL3–18 was transiently transfected into A10 cells. Insulin was added for 30 min (I30) or 60 min (I60) or cells were not treated with insulin (I0). Total RNA was isolated and RT-PCR was performed using primers to ßII-SA. C, The truncated minigenes pSPL3–19, pSPL3–20, and pSPL3–22 were transiently transfected into A10 cells. Insulin was added for 30 min (I30) or 60 min (I60), or cells were not treated with insulin (I0). Total RNA was isolated and RT-PCR was performed using primers to ßII-SA. ß-Actin was used as an internal control. The experiments were repeated at least two times to ensure reproducible effects of insulin.

 
Further deletion of an additional 177 nucleotides, eliminating the AU-rich element sequence but maintaining the SRp40 binding site in pSPL3–19 minigene, also preserved insulin-regulated activation of splice sites (Fig. 7BGo). Interestingly, however, splice site II was activated only after 60 min of insulin treatment. It could be hypothesized that the AU-rich element was involved in secondary structure formation facilitating splice site selection. Also, the splicing factor hnRNP A1 binding site is downstream of the SRp40 site (see Fig. 7AGo). This may suggest that elimination of the AU-rich element may have enabled hnRNPA1 to bind to an intronic splicing silencer, thereby decreasing the utilization of splice site II. Further studies are indicated to clarify the role of hnRNP A1.

Minigene pSPL3–20 truncated at the SRp40 site, destroying a portion of it, showed splice site I activation within 30 min; however, splice site II was not used even at 60 min (Fig. 7BGo). This indicates that SRp40 is a key factor in early insulin regulation of splice site selection of PKCßII exon and is essential for splice site II activation.

The removal of the SRp40 binding site and the proximal sequences in minigene pSPL3–22 to a minimum of 39 nucleotides of the 5'-intronic sequences resulted in only 20% utilization of splice site I even at 60 min after insulin treatment. This emphasizes the importance of cis-elements flanking the 5'-splice site in PKCßII exon inclusion and 5'-splice site selection by insulin (Fig. 7BGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The regulation of alternative splicing by hormonal or metabolic stimuli is emerging as a highly intriguing area of RNA processing. Various mechanistic studies have been carried out to determine tissue-specific or developmentally specific alternative splicing (41). Regulation of alternative splicing by signal transduction pathways shows promises of a leading candidate for gene therapy in diseases [see review by Stamm (42)]. The complexity of the entire mammalian PKCß gene makes it difficult to identify the cis-elements involved in insulin-regulated splicing of PKCßII mRNA. The development of a heterologous minigene provides a valuable tool for analyzing the sequences on the pre-mRNA directing insulin-stimulated PKCßII exon inclusion. The pSPL3–32 minigene, containing the entire exon and the flanking portions of 3'- and 5'-introns, mimicked the endogenous insulin response of PKCßII alternative splicing including the regulation of multiple splice sites in five distinct cell types: skeletal muscle, vascular smooth muscle cells, hepatoma cells, preadipocytes, and fibroblasts. These are all derived from tissues in which PKCßII has been shown to have a role in glucose utilization or storage (43, 44, 45, 46, 47). The minigene elucidates insulin-regulated splicing and splice site activation in complex scenarios where observation of the endogenous phenomena is difficult due to multiple levels of posttranscriptional regulation (15).

The deletion analysis of the heterologous minigene pSPL3–32 revealed that insulin predominately regulated splice site I and II selection via adjacent downstream intronic elements. The regulatory elements activated in response to insulin stimulation resided within the 5'-intron flanking the PKCßII exon because deletion of the 3'-intronic sequence (pSPL3–17 minigene construct) had no effect on insulin-stimulated splice site selection. However, deletion of the SRp40 binding site by mutation or truncation of the minigene resulted in altered use of splice sites I and II. Here, the increased expression of SRp40 also mimics insulin. Insulin transcriptionally regulates SRp40 levels (18) in addition to increasing its phosphorylation (28). Additional work is required to elucidate the precise binding mechanisms of other specific factors associated with insulin-regulated alternative splicing; however, we have identified specific regions and elements that confer insulin-responsive splice site selection to the PKCßII mRNA.

A PI-3 kinase signal transduction pathway appears to be closely associated with insulin-regulated splice site selection. Here, we have shown that a predominantly metabolic signaling pathway regulates 5'-splice site selection for an exon, PKCßII, presumably activating a PI3-kinase/Akt2 kinase phosphorylation pathway. Phosphorylation of SRp40 by Akt2 kinase (34) and the activation of splice site selection by CA-Akt2 kinase of the heterologous minigene further emphasize the importance of this phosphorylation cascade in fine tuning of cellular events. Other signaling pathways associated with regulated splicing include the ERK phosphorylation of splicing factors (48) and calcium activation of Ca2+/calmodulin-dependent protein kinase, calmodulin-dependent protein kinase IV (49), and other calcium-dependent kinases (50).

The regulation of alternative splicing of PKCßII by insulin presents another scenario for regulated alternative splicing, which is distinct from the developmental and tissue-specific models that have classically been shown to be regulated. Here, a hormone, the levels of which change dramatically in response to extracellular glucose concentrations, is shown to regulate exon inclusion and splice site selection in target tissues expressing the insulin receptor signaling pathways and factors involved in splicing. Interestingly, the product, PKCßII, is involved in insulin signaling (11) and may also be required to terminate the signal sequence by phosphorylating the insulin receptor (16, 51).

In conclusion, a model system has been developed to elucidate the mechanisms involved in hormonally regulated alternative splicing in a number of cell types independent of developmental stages. The results define an intronic sequence that opens the topic of how splice site selection is controlled by metabolic signaling pathways in normal physiology and disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
Rat L6 skeletal myoblasts (obtained from Dr. Amira Klip, The Hospital for Sick Children, Toronto, Canada) were grown in {alpha}-MEM supplemented with 10% fetal bovine serum to confluence. Cells were fused by changing media to {alpha}-MEM supplemented with 2% fetal bovine serum for 4 d postconfluence with media changed daily. The extent of differentiation was established by observation of multinucleation of 85–90% of cells. Myotubes were incubated in {alpha}-MEM with 0.1% BSA for 6 h and placed in PBS with 0.1% BSA just before treatment with insulin.

The vascular smooth muscle cell line (A10, ATCC CRL 1476), derived from rat aorta, was grown in DMEM (with 5.5 mM glucose) containing 10% fetal bovine serum, 100 U penicillin G, and 100 µg/ml streptomycin sulfate, at 37 C in a humidified 5% CO2-95% air atmosphere in either six-well plates or 100-mm plates.

The human hepatoma cell line, HepG2, was grown in ATCC MEM supplemented with 10% fetal bovine serum, 100 U penicillin G, and 100 µg streptomycin sulfate/ml, at 37 C in a humidified 5% CO2-95% air atmosphere.

Rat hepatocytes were isolated as described previously (52). Briefly, liver perfusions to isolate hepatocytes were performed on ad libitum fed Sprague Dawley rats using collagenase (53). Isolated hepatocytes were purified on Percoll gradients (54). Purified viable hepatocytes were seeded (2 x 106 cells/2 ml per 60-mm dish) in Waymouth’s 752/1 medium containing 0.2% BSA (wt/vol) 100 U Penicillin G, and 100 µg/ml streptomycin sulfate, 50 µg/ml gentamicin (referred to as complete) on rat tail collagen-coated petri dishes and allowed to attach for 3 h at 37 C in an humidified chamber containing 5% CO2-95% air atmosphere. After plates were washed with 0.2% BSA/Hanks’ balanced salt solution to remove nonadherent cells, cells were reincubated in complete Waymouth’s medium containing 0.1 nM insulin overnight. After cells were washed three times with 0.2% BSA/ Hanks’ balanced salt solution, cells were reincubated in complete Waymouth’s for 1 h and then incubated with and without 100 nM insulin for 10, 30, and 60 min. At each time point, cells were harvested and total RNA was isolated in TRIZOL reagent (Tel-Test, Inc., Friendswood, TX) as described by the manufacturer.

The 3T3 preadipocytes obtained from the American Type Tissue Culture repository, ATCC (Manassas, VA) were maintained and passaged as preconfluent cultures in DMEM (Sigma Chemical Co., St. Louis, MO) high glucose with 10% newborn calf serum (Sigma) at 37 C and 8% CO2. At confluence, cells were differentiated by incubation in medium containing 25 mM glucose, 10% fetal bovine serum, 1 µg/ml insulin, 1 mM dexamethasone, and 0.5 mM isobutyl-1-methylxanthine. After 4 d the medium was changed to DMEM, 25 mM glucose, and 10% fetal bovine serum. Differentiated adipocytes were used for the experiments 12 d after initiation of differentiation and were placed in a serum-depleted medium for 3 h before insulin stimulation.

The murine embryonic fibroblasts, MEF cells [obtained from M. Birnbaum (55)], were grown in DMEM, high glucose, supplemented with 15% FCS, 2 mM L-glutamine, and penicillin (100 U)-streptomycin (100 µg/ml) and kept at 37 C in a humidified 5% CO2-95% air atmosphere.

Construction of the Heterologous pSPL3-PKCßII Splicing Minigene
The pSPL3 vector (56) contains the HIV-1 tat exons with SD site and the SA site and the intervening intron with a multiple cloning site. Upon transfection, the product is transcribed using the simian virus 40 early promoter. Packaged {lambda}RG32, a rat genomic clone containing the PKCßII exon and its flanking intronic sequences (9), was digested with SacI and XbaI and separated on a 0.7% agarose gel. The PKCßII exon with a portion of its flanking intronic sequences (1000 bp on the 5'-end and 1200 bp on the 3'-end) was then excised and purified using the Qiaquick Gel extraction kit (QIAGEN, Chatsworth, CA). The splicing vector pSPL3 was digested with SstI within the multiple cloning site and downstream NheI, removing the cryptic 5'-splice site. PKCßII with its flanking introns was cloned into the pSPL3 vector between the SD and SA sites and called pSPL3–32. Positive clone in the correct orientation was confirmed by restriction digestion and sequencing.

For the construction of pSPL3–17, {lambda}RG32 was digested with EcoRI (within the PKCßII exon) and XbaI. A 3'-splice site sequence (with one mismatch to the consensus sequence), which the exon can utilize for splicing, was introduced in the primer. The segment thus obtained was cloned into the splicing vector pSPL3, which was digested with EcoRI and XbaI. Positive clone was confirmed by restriction digestion and sequencing.

Deletion constructs pSPL3–18, pSPL3–19, pSPL3–20, and pSPL3–22 were generated by cloning the PCR products into the splicing vector pSPL3, digested with EcoRI and XbaI. The primer pairs used included the EcoRI site and the XbaI site in its design to facilitate cloning in the correct orientation. Forward primer for all the constructs was 3'-CAGGAAGTCATCAGGAATAT-5. It also contained a 3'-splice site sequence with one mismatch which the exon can utilize for splicing. The reverse primers for pSPL3–18 were 3'-CGTCTAGACTATGAGAGGAAGTGCTTTT-5'; for pSPL3–19, 3'-TCTCTAGAGGGCAAAGCAGCCATATACT-5'; for pSPL3–20, 3'-GCCATATAGCTAGCTCAAGCCAAGCTCCCAGCCG-5'; and for pSPL3–22, 3'-CACGGAGCTAGCTTGGCAATGGAAAAGGAAAA-5'. Positive clones were confirmed by restriction digestion and sequencing.

Lipofectamine Transfections
Cells were grown in either 35-mm or 100-mm plates. The expression vectors were transfected in serum-free medium with Lipofectamine. Lipofectamine reagent was purchased from Invitrogen, Life Technologies (San Diego, CA). The Lipofectamine-DNA complex was incubated at room temperature for 20 min, diluted with the serum-free transfection medium, and added to the cells. The cells were incubated at 37 C for 4 h and then incubated in the growth medium 37 C for 18 h.

RT-PCR Analysis
Total RNA was isolated from cells using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to manufacturer’s instructions. One microgram was used to synthesize first-strand cDNA using an Oligo(dT) primer and Superscript II Reverse Transcriptase (Life Technologies Pre-amplification Kit, Life Technologies, Gaithersburg, MD). The sense primer for ßII exon was 5'-CCCGAAGTCAAGAGCTAAGTAG-3'; for SD was 5'-TCTCAGTCACCTGGACAACC-3'; and antisense primer for SA was 5'-CCACACCAGCCACCACCTTCT-3'. Sense and antisense primers for ß-actin (catalog no. 5402–3, CLONTECH Laboratories, Inc., Palo Alto, CA) were used to normalize for total RNA. After 30 cycles of amplification in a Biometra Trioblock thermocycler (ßII-SA: 94 C, 1 min; 60 C, 1 min; and 72 C, 3 min; SD-SA: 94 C, 1 min; 58 C, 1 min; and 72 C, 3 min; and ß-actin: 94 C, 1 min; 58 C, 1 min; and 72 C, 3 min for 25 cycles) 5% of products were resolved on 6% PAGE gels and detected by silver staining, or 10% of products were resolved on 1.2% agarose gels and detected by ethidium bromide staining. The PCR reaction was optimized for linear range amplification to allow for quantification of products. Densitometric analyses of the bands were done using the Scan Analysis Software (Biosoft, Cambridge, UK).

Southern Blot Analysis
The PCR product was transferred to Hybond N nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) and cross-linked by baking at 80 C in a vacuum oven for 2 h. Blots were hybridized overnight at 42 C with PKCßII cDNA (labeled with [32P]dCTP by nick translation) in a hybridization buffer containing 5x SSC, 5x Denhardt’s solution, 0.5% lauryl sulfate, and 50% formamide. Membranes were washed with high-stringency conditions and exposed to an imaging screen. Blots were then quantitated using the PhosphorImaging system (Molecular Dynamics, Sunnyvale, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. Jin Q. Cheng (University of South Florida, Tampa, FL) for the CA-Akt2 constructs and Dr. M. Birnbaum (Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA) for the embryonic fibroblasts. The {lambda}RG32 was obtained from Dr. Yoshitaka Ono, Takeda Chemical Industries, (Osaka, Japan). We thank Daniel Mancu for technical support.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant NIDDK 54393 (to D.R.C.) and the Department of Veteran’s Affairs Medical Research Service (D.R.C.).

Abbreviations: CA-Akt2, Constitutively active Akt2 kinase; hnRNP, heterogeneous nuclear riboprotein; PI3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; SA, splice site acceptor; SD, splice site donor; SR proteins, serine/arginine-rich proteins.

Received for publication October 9, 2003. Accepted for publication January 19, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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