Inhibition of Expression of Protein Kinase C a by Antisense cDNA Inhibits Phorbol Ester-mediated Arachidonate Release*

A major unresolved issue in the area of signal trans- duction relates to the role of particular isoforms of protein kinase C (PKC) in mediating cellular responses subsequent to activation of that enzyme. We have addressed this issue by the use of antisense technology. We have stably transfected Madin-Darby canine kid-ney cells with antisense PKCa, PKC/3, or both PKCa and -6 cDNAs. The transfected cDNA was integrated and expressed. We have isolated cells in which expression of PKCa is inhibited. In cells transfected with antisense PKCa or both PKCa and -6, phorbol ester-stimulated release of arachidonate and its metabolites was inhibited, whereas in cells transfected with antisense PKCB cDNA alone, phorbol ester-stimulated ar- achidonate release was not significantly different from control cells. We thus demonstrate the use of a novel technique to inhibit PKC isoform expression. We show that inhibition of expression of PKCa causes a loss in phospholipase Az-mediated arachidonate release. An- tisense-inhibited expression of PKC isoforms may provide a useful approach to define additional functions of particular PKC isoforms.

8 To whom correspondence should be addressed. Tel: 619-534-The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; kb, kilobase pair(s); CKP, antisense PKC cDNA; CMV, cytomegalovirus; AA, arachidonic acid; PLA2, phospholipase AP; cPLA2, cytosolic phospholipase AP; MDCK, Madin-Darby canine kidney. encoded by individual genes. The a, p, and y forms of PKC were the first to be cloned (1) and to be characterized biochemically (5). (PKCP actually consists of two forms that are alternatively spliced variants of a single gene.) PKCa, -D, and -7 are all phospholipid-, diacylglycerol-, and calcium-dependent. However, subtle differences in their cofactor dependences, their susceptibility to down-regulation, and their tissuespecific expression has led to the speculation that different isoforms perform specific functions. The more recently described 6, t, 5; q, and L PKC isoforms are also dependent on phospholipid and diacylglycerol for their activation, but lacking the putative calcium binding site expressed in the regulatory region of PKCa, -P and -7, they are calcium-independent and show distinct in vitro substrate specificities (6).
It has been difficult to demonstrate specificity of function of a single isoform in uiuo. As isoform-specific inhibitors of PKC are not available, typical experimental paradigms used to implicate a particular form of PKC are its translocation and/or phorbol ester-induced down-regulation (e.g. . Such approaches provide circumstantial evidence implicating differential activation of PKC isoforms in a specific process. However, the down-regulation paradigm has the complicating factor of "activation" of the kinase being a prerequisite for its down-regulation (4). Additionally, recent evidence suggests that PKC translocation may not be required for its activation (10). Overexpression of PKC isoforms has recently been used to implicate them in particular functions (e.g. Refs. 11 and 12). However, in these systems, substrate availability, ligand receptor number, or other regulators may critically limit responsiveness of the system. In an effort to circumvent these problems, we have developed a strategy to isolate stably transfected cells deficient in a specific isoform in order to determine whether a specific PKC-mediated response is altered.

Methods
Plasmid Constructs-All constructions were made according to standard procedures (15). 1.3-and 1.7-kb EcoRI fragments containing PKCa or PKC& respectively, were isolated from the pUC host vector and subcloned into the mammalian expression vector pCMV-1 in the antisense orientation using ClaI and XbaI (PKCa and BglII and ClaI (PKCP). This yielded antisense PKC (CKPa and CKPP) preceded by the CMV promoter and followed by the human growth hormone termination and polyadenylation signals (Fig. 1). All constructs were confirmed by sequence analysis.
Celk and Transfections-MDCK-Dl cells were cultured as previously described (16). Cells (approximately 1 X lo6 cells/lOO-mm dish) were co-transfected with pCMV expression vectors containing antisense PKCa and/or PKCp and the neomycin resistance gene (RC-CMVNeo, In Vitrogen) using calcium phosphate coprecipitation (15). Control cells were transfected with Rc-CMVNeo only. A 5-fold mass excess of PKC cDNA over the selectable plasmid was used in transfections. For post-transfection, the cells were plated in medium containing 800 pg/ml G418 (GIBCO/BRL). Colonies of resistant cells appeared after -18 days and were picked for expansion at 21 days. Stock cultures of these cells were routinely grown in the presence of selective pressure. Southern Blot Analysis-Genomic DNA from the transfectants and parental cells was prepared by the method of Herrmann and Frischauff (17). Genomic DNA (30 pg) was digested with EcoRI, separated on a 1% agarose gel, and, following denaturation and neutralization, was transferred to nitrocellulose.
The blots were probed with specific PKCa and PKCp cDNA probes random hexamer-primed (U. S. Biochemical Corp. random priming kit) to a specific activity >1 X IO9 cpm/pg. Bound probe was detected by autoradiography. Southern analyses were performed twice on each of the various cell lines.
Northern Blot Analysis-Total RNA was isolated from stable transfectants and wild type MDCK-Dl cells by the method of Chomczynski and Sacchi (18). 30 fig of total RNA from each cell type was electrophoresed on a 1.2% formaldehyde gel and transferred to nitrocellulose. Loading of approximately equivalent quantities of RNA per lane was verified by ethidium bromide staining of the gel. The blots were probed with PKCa and PKCp random hexamer-primed cDNA probes, as described above. Northern analyses were performed twice on the transfectants.
Western Blot Analysis-Lysates from transfectants and parental cells were prepared as described elsewhere (16) substituting 1% Nonidet P-40 for Triton X-100 in the lysis buffer. Equivalent amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis before transferring to nitrocellulose and probing with PKC isoformspecific antisera (19). Bound antibody was detected using lZ5I-Protein A. Western blotting experiments were carried out at least three times on the transfectants.
PKC Activity-PKC activity in lysates from the transfectants and wild-type cells was measured by determining diacylglycerol and phospholipid-dependent histone phosphotransferase activity after partial purification of the kinase by DEAE-Sephacel chromatography (16).
Arachidonate Release-PMA-stimulated release of arachidonic acid and arachidonate metabolites (AA) from wild type and transfectants was measured by a method similar to that previously reported from our laboratory (20). Cells were labeled with [3H]arachidonate (0.3 pCi/ml of medium) for 18 h. After gently washing with Dulbecco's modified Eagle's medium, 20 m M HEPES, 0.05% bovine serum albumin (incubation medium), the cells were then treated with various concentrations of PMA (0-1 p~) in incubation medium for 60 min at 37 "C. 3H released into the extracellular medium was determined by liquid scintillation counting.

RESULTS AND DISCUSSION
To date it has been difficult to ascribe specific functions to PKC isoforms with certainty. A novel approach to this end is to selectively inhibit expression of the kinase and then to determine if specific responses are altered. Antisense technology provides a unique approach to this problem. The basis of this technique is that expression of native proteins can be inhibited by hybridization of native mRNA to complementary nucleotide sequences.
In preliminary experiments we treated MDCK cells in culture with high concentrations of oligodeoxynucleotides generated complementary to unique 18-nucleotide sequences in the 5' region of PKC. Although the initial data demonstrating decreased i n vitro PKC activity relative to sense-treated controls were promising, attempts to optimize the system by establishing the kinetics of uptake of polynucleotide kinase-labeled oligodeoxynucleotides revealed extensive intracellular digestion of oligodeoxynucleotides within 12 h of addition to the extracellular medium. This approach is probably more appropriate for inhibition of expression of proteins with relatively short half-lives (e.g. Refs. 21 and 22).
Thus, we decided to use an alternative strategy: stable transfection of PKC isoform cDNA. Cells were cotransfected with cytomegalovirus promoter-based expression vectors containing the neomycin resistance gene and either antisense PKCa (pCMVCKPa) or antisense PKCp (pCMVCKPp), or both (pCMVCKPa + pCMVCKPP). Control cells were transfected with the neomycin resistance gene only. Stable transfectants were isolated on the basis of colony formation in 800 pg/ml G418. Having isolated resistant colonies, we then investigated integration and expression of the transfected genes using Southern and Northern analysis, respectively.
Integration of transfected DNA into the host MDCK-Dl genome was assayed by Southern blotting. As shown in Fig.   1, panel A, probing of EcoRI-digested genomic DNA from the wild-type parental and transfected cells with a PKCa cDNA probe revealed hybridization to approximately 7.0and 5.5kb fragments in all cells (i.e. wild-type, CKPa, CKPP, and cells transfected with the neomycin resistance gene alone (neo')). In cells transfected with the pCMV CKPa construct we detected an additional fragment at 1.3 kb, thus indicating incorporation of CKPa into the genomic DNA of the appropriate transfectants.
All the transfectants harboring the CKPa construct appear to do so a t approximately the same copy number. Probing EcoRI-digested genomic DNA with a PKCD probe revealed strong hybridization to a 7.0-kb fragment and much weaker hybridization to 6.5-and 5.8-kb fragments in all cells, whereas in cells transfected with CKPp an additional signal was detected at 1.7 kb, indicative of the integration of CKPP into the genomic DNA of the transfectants (Fig. 1, panel B ) .
Having determined that the transfected genes had been integrated into the host cell genome, we used Northern analysis to assess transcription of the DNA. The PKCa and PKCp probes hybridized to transcripts of approximately 4.4 kb in all cells (wild type and transfectants). In CKPa-and CKPPtransfected cells additional transcripts were detected a t -1.9 and 2.3 kb, respectively (Fig. 2, A and B ) . This corresponds to the anticipated size of transcripts produced by the transfected genes. The size of the major native transcript detected agrees well with that reported by others (10,14,15). Densitometric analysis of the Northern blots showed an approximately 5-fold excess of CKPa mRNA over the endogenous PKCa transcript and a 1-3-fold excess of CKPp mRNA over the native PKCp message.
Given the demonstrated integration and expression of the transfected genes, we then addressed whether expression of the antisense RNA for the PKC isoforms was capable of inhibiting translation of the proteins. T o investigate this we used Western blotting with isoform-specific antisera (19).  (3) and from cells transfected with the neomycin resistance gene alone (Neo" as indicated were separated on a 1% agarose gel, transferred to nitrocellulose, and probed with PKCa or PKCP cDNA probes (panels A and B, respectively) random hexamer-primed to a specific activity >1 X lo9 cpm/pg. The blot was prehybridized by incubation at 42 "C in 5 X SSPE, 5 X Denhardt's, 50 pg/ml salmon sperm DNA, 50% formamide for 2-4 h. The blot was incubated overnight with 1 X lo6 cpm of probe/ml of hybridization solution at 42 "C and then washed to relatively high stringency before autoradiography. The migration of a 1-kb ladder (GIBCO/BRL) is shown a t the Left side of each blot. Arrowheads indicate the anticipated sizes of the integrated genes. In cells cotransfected with CKPa equivalent hybridization to that shown in lanes 5-10 was observed. neo'transfectants as indicated were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The blot was probed with PKCa-specific antisera and bound antibody detected using '*'I-Protein A. Purified PKC was used as a positive control in these experiments, and its migration is indicated at the right-hand side of the blot. unable to show definitively that the antisense PKCp gene, despite its integration and expression ( Figs. 1 and 2), inhibited expression of the protein by Western blotting.
Of the stably transfected cell lines established, no false positives were obtained in screening the co-transfectants for integration and expression of the transfected genes (from 20 expanded colonies).' However, despite continuous selective pressure on the transfectant cell lines (i.e. numerous passages), expression of the antisense genes was substantially reduced with time. This was evident by a considerably weaker signal in Southern and Northern blots relative to the endogenous gene or transcript, respectively. Additionally, decreased expression of the proteins was not detected in Western blots of extracts from stably transfected cells of higher passage number. The data presented in this paper were obtained from transfectants between passages 5 and 20.
In addition to determining expression of the PKC isoforms in Western blots, we measured total PKC activity in cell lysates using histone as a substrate. In all antisense PKC cDNA transfected cells a decrease in PKC activity was observed (Fig. 4). This decrease in enzyme activity showed variability between the cell lines established from transfection with the various cDNA vectors CKPa, CKPP, or CKPa + CKPp and did not correlate exactly with the relative magnitude of the decrease in protein expression observed in the immunoblotting experiments. A significant ( p < 0.005) decrease in histone phosphotransferase activity was observed in CKPa and CKPa + CKPp transfectants compared with either the wild-type or neo' controls. It is likely that some of the residual PKC activity results from expression of non-calciumdependent isoforms, in particular PKC, in MDCK cells (23). In the CKPp transfectants PKC activity was reduced to approximately 70% of the wild type. This relatively modest decrease (insignificant compared with the neo' cell lines) may reflect the relative amount of the a and / 3 present in the cells In a single transfectant cell line, positive for both integration and expression of CKPa, no decrease in PKCa was observed. was determined in wild-type cells and CKPa, CKPP, CKPa + CKBP, and neo' transfectants. Cells were labeled with 3H-labeled AA overnight, the labeling medium was removed, and, after washing, cells were incubated with 50 nM PMA for 60 min, and the release of 3Hlabeled arachidonate and its metabolites into the extracellular medium was determined. Results shown are the mean & S.E. of four experiments; each experiment was performed in duplicate. as both have similar K,,, values for histone phosphorylation (6). However, why the histone phosphotransferase activity in the CKPa + CKPp transfectants is not lower than in the CKPa transfectants alone is unclear. On the basis of decreased specific isoform expression and decreased PKC activity, CKPa, CKPP, and CKPa + CKPp cell lines were selected for further experiments.
There are several possible mechanisms whereby expression of antisense RNA can block expression of a target gene (e.g. Ref. 24). These include accelerated turnover of the senseantisense duplex, which would decrease abundance of target mRNA, decreased transportation of the sense-antisense duplex from the nucleus, inhibition of mRNA binding to the ribosome with subsequent inhibition of protein translation, and prolongation of ribosomal pausing during translation leading to a reduction in protein translation. In our system accelerated turnover of the duplex is unlikely to account for the decreased expression of PKC in the antisense transfectants as we do not observe a difference in mRNA for the endogenous PKC genes between the transfectants and the wild-type cells. Our observation that expression of excess antisense RNA inhibits expression of the protein may be due to any of the alternative potential mechanisms described above or to a combination thereof.
The major objective of this work was to determine whether selective inhibition of a PKC isoform alters a specific biological response: PMA-stimulated release of arachidonate and arachidonate metabolites. Arachidonate formation is the ratelimiting step in the synthesis of several potent biological mediators such as prostaglandins and leukotrienes. Previous work from this Iaboratory has strongly suggested that AA release in response to phorbol esters and to hormones is due to phospholipase Az activation in this system (25). In CKPa and CKPa + CKPp transfectants, we observe negligible AA release in response to PMA, whereas in parental cells and neo' cells a 4-fold increase was observed at 50 nM (Fig. 4). In CKPp transfectants, a 3.5-fold increase in AA release was observed in response to 50 nM PMA and thus was not significantly different from that of the control cells. The modest decrease in AA release from the CKPB transfectant may reflect slight inhibition of PKCa expression in these transfectants due to the high sequence homology between the isoforms; however, as we do not see an additive effect on inhibition of AA in CKPa + CKPp uersus CKPa transfectants we discount this possibility. These data substantiate work using translocation and down-regulation paradigms to implicate PKCa, but not PKCP, in PMA-and hormone-stimulated AA release (13). As a further control for the effects of antisense PKC transfection on AA release, MDCK cells were stably transfected with sense PKCa cDNA engineered so as not to be translated into protein (lacking the carboxyl-terminal 36 amino acids). In these experiments PKCa protein expression was unchanged, and PMA-stimulated arachidonate release was similar to that observed in wild-type cells.
A cytosolic PLAZ (cPLA2), which is responsible for hormone-stimulated arachidonate release, has recently been described (26-29). There are several possible mechanisms whereby PKCa might effect activation of cPLA2. These are: regulation of cPLAz-activating proteins such as phospholipase activating protein (30) or lipocortin (31), alterations in transmembrane calcium flux (32), G protein coupling (33), or direct regulation through phosphorylation of the phospholipase (34). We have investigated possible phosphorylation of cPLA2 using PLAz-specific antisera. Immunoprecipitation of cPLAz from cells labeled with 32P and stimulated with phorbol ester or hormone revealed no detectable difference in phosphorylation of the protein relative to basal conditions (data not shown). In other systems the stoichiometry of cPLAz phosphorylation and AA release suggest that phosphorylation is a necessary but insufficient stimulus for maximal activation; elevations in intracellular calcium are also required. This is compatible with data showing a synergistic effect of PMA on A23187-mediated AA release (17) and the hypothesis that the ability of an agonist to both activate PKC and to elevate intracellular calcium determines its efficacy in releasing AA (35).
In summary, we have used a novel technique to inhibit expression of a specific PKC isoform. We have shown that inhibition of expression of PKCa causes a loss of PLA2mediated AA release in response to PMA. The use of antisense to inhibit expression of PKC isoforms may provide a useful approach to study differential activation and function of PKC isoforms.