Department of Medicine (C.A.P.), Division of Metabolism, Endocrinology and Nutrition, University of Washington School of Medicine, Seattle, Washington 98195-6426; Department of Medicine and of Biochemistry, Biophysics and Genetics (N.M., A.G.-H.), Program in Molecular Biology and Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262; and Department of Molecular Cell Physiology (Y.A.), Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan
Address all correspondence and requests for reprints to: Cheryl A. Pickett, M.D., Ph.D., Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, Box 356426, University of Washington School of Medicine, 1959 Northeast Pacific Street, Seattle, Washington 98195-6426. E-mail: cpickett{at}u.washington.edu.
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ABSTRACT |
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INTRODUCTION |
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Given the known direct effect of phorbol esters on protein kinase C (PKC) and data demonstrating that both TRH and EGF can stimulate PKC via activation of phospholipases and subsequent release of PKC activators, members of this kinase family are likely candidates for regulating the effects of EGF and TRH on PRL gene transcription. Several previous studies have indirectly examined the question of convergent pathways in mediating TRH, EGF, and phorbol ester effects on PRL synthesis and secretion; however, none have demonstrated a clear role of PKC in mediating the EGF effect on PRL gene regulation. Furthermore, none have examined the role of specific PKC isozymes in EGF- and TRH-mediated PRL gene regulation.
The PKC family of serine/threonine protein kinases plays critical roles in many signal-transducing pathways in the cell (for review, see Refs. 8 and 9). Eleven distinct PKC isozymes have been identified in mammalian cells. These have been divided into three subfamilies: conventional or Ca2+-dependent cPKCs (cPKC, ßI, ßII, and
), novel or Ca2+-independent nPKCs (nPKC
,
,
, and
), and atypical aPKCs (aPKC
and
). The cPKCs require calcium, diacylglycerol (DAG) or exogenous phorbol esters, and the cofactor phosphatidyl-L-serine (PS) for full activation. Novel PKCs also require DAG (or TPA) and PS for activation; however, calcium does not enhance activation. Atypical PKCs are not activated by either DAG or TPA and are unresponsive to calcium (9). Phosphoinositide products of the phosphoinositide 3-kinase have also been shown to activate the novel and the atypical PKCs.
GH4C1 cells express PKC transcripts for cPKC, cPKCßII, nPKC
, nPKC
, nPKC
, and aPKC
, whereas transcripts for cPKC
and nPKC
have not been detected (10). nPKC
is the most abundant isozyme in GH4-derived pituitary cells (10).
In vitro, phorbol esters (TPA) produce a prolonged activation of PKC isozymes via a non-Ca2+-dependent mechanism involving competitive interaction at the DAG binding site (11). In vivo, phorbol ester activation of PKCs has been inferred by evidence of down-regulation of PKC expression and/or by demonstration of redistribution of PKCs from the soluble to particulate subcellular fractions (for review, see Ref. 12). TPA is not an isozyme selective agonist in that it appears to activate the kinase activity of multiple PKC isozymes in most cell types and can down-regulate expression of all the PKC isozymes except the atypical PKCs and
. The various isozymes do, however, have different susceptibilities to down-regulation by TPA, with cPKCßII and nPKC
more susceptible than PKC
and PKC
(13, 14). In GH4C1 cells, cPKC
, cPKCßII, nPKC
, nPKC
, and nPKC
have all been reported to be translocated to the particulate fraction in response to TPA (14, 15, 16). In GH4 pituitary cells, TPA treatment results in enhanced PRL secretion (17) and PRL gene transcription (4, 18); the latter effects can be blocked with staurosporine and sphingosine at concentrations that are selective for PKC (20).
TRH-mediated activation of PKC occurs as a result of TRH receptor coupling to G protein -subunits, resulting in activation of phospholipase C ß1 (21). Subsequently, phosphatidylinositol 4,5-bisphosphate hydrolysis occurs with increases in the levels of two PKC cofactors, DAG and Ca2+ (1, 22). TRH appears to activate the kinase activity of multiple PKC isozymes as indicated by redistribution studies (10, 14, 15, 23). However, TRH specifically down-regulates nPKC
and not cPKC
, cPKCßII, nPKC
, or aPKC
expression (10). Previous data have suggested that nPKC
is involved in the regulation of TRH-induced PRL secretion (10). Overexpression of nPKC
under either stable or transient conditions resulted in increased basal PRL secretion and enhanced the TRH response in GH4C1 cells in these studies. In contrast, transient overexpression of PKCs
, ßII, and
did not significantly affect either basal or TRH enhanced PRL secretion. A role for nPKC
in mediating TRHs effect on PRL gene transcription has also been inferred from the temporal correlation between TRH-stimulated DAG accumulation, nPKC
down-regulation, and decreased PRL mRNA synthesis (15, 24).
EGF binding to the EGF receptor tyrosine kinase at the cell membrane results in the phosphorylation and activation of several protein substrates, including Grb/Sos/Ras, phospholipase C-1, phosphatidylinositol 3-kinase, and signal transducers and activators of transcription proteins (25). These substrates of the EGF receptor tyrosine kinase then appear to transmit the EGF signal to various intracellular compartments via phosphorylation of downstream kinases or by other signal transduction mechanisms. We have previously demonstrated that EGF stimulates rPRL gene transcription in GH4T2 cells via a pathway that is separate and antagonistic to the Ras pathway. Furthermore, this pathway does not appear to require Raf kinase, MAPK, or c-Ets 1 (26, 27). Although several indirect lines of evidence have implicated PKCs in EGF-mediated effects on PRL gene regulation and secretion, this has not been extensively studied. Furthermore, previous studies in which prolonged TPA treatment failed to inhibit subsequent EGF-stimulated PRL transcription have been interpreted to indicate that PKCs are not required for the EGF response (28).
In the studies described here, we have examined the effect of several selective and specific PKC inhibitors on the EGF- and TRH-induced activation of the rPRL promoter. We have also examined the effect of PKC overexpression in both stable and transiently transfected GH4C1 cells on TRH-, TPA-, and EGF-induced rPRL promoter activity. Finally, we have investigated the effect of overexpression of PKC isozymes
, ßI, ßII,
,
,
,
,
, and
, and the effect of transient expression of kinase inactive mutants of several of these isozymes on the EGF and TRH responses. Our data indicate that staurosporine, bisindolylmaleimide I, and Calphostin C inhibit both EGF and TRH activation of the rPRL promoter. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of DAG than are EGF effects. G0 6976, a selective inhibitor of Ca2+-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Significant inhibition of both EGF- and TRH-mediated activation was observed with Rottlerin, a specific inhibitor of the novel nPKC
. Neither stable nor transient overexpression of nPKC
produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Overexpression of nPKC
produced a modest enhancement of the EGF response, whereas overexpression of other PKC isozymes including nPKC
failed to significantly enhance either the EGF, TRH, or TPA responses. Collectively, these data suggest that several PKCs are involved in both EGF and TRH effects and that only a relatively small proportion of activated kinase is necessary to mediate these responses and/or that the mediator(s) downstream of the PKCs rather than PKCs themselves are limiting in regards to transcription of the rPRL promoter.
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RESULTS |
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In Fig. 1, data from experiments utilizing these inhibitors in EGF- and TRH-treated GH4T2/GH4C1 cells are depicted. As shown in Fig. 1A
, EGF induced a 5-fold stimulation and TRH a 3-fold stimulation of rPRL promoter activity in the absence of staurosporine. With increasing concentrations of staurosporine, present before the addition of EGF or TRH, a dose-dependent inhibition of rPRL promoter activation was observed. When cells were treated with bisindoylmaleimide I (Fig. 1B
), however, similar effects upon both EGF- and TRH-stimulated rPRL promoter activity were observed. Bisindoylmaleimide I produced inhibition of both TRH- and EGF-stimulated rPRL promoter at nanomolar concentrations, suggesting that these factors utilize a PKC-dependent pathway in regulating rPRL gene transcription. Calphostin C (Fig. 1C
) also produced a dose-dependent inhibition of both EGF- and TRH-activated rPRL promoter activity, implicating a requirement for DAG binding. However, the concentration of Calphostin C required to inhibit EGF-mediated activation of the rPRL promoter was approximately 10-fold that producing inhibition of the TRH effect and in the 0.1 µM range. Finally, Go 6976 produced a modest inhibition of EGF-induced rPRL promoter at concentrations equal to or greater than 10-8 M, whereas TRH was not significantly inhibited at concentrations up to 10-6 M (Fig. 1D
). None of these agents significantly inhibited cytomegalovirus (CMV) or Simian virus (SV) promoter activity. Only staurosporine and Calphostin C produced any significant inhibition of basal rPRL promoter activity and then only at the highest (1 x 10-6 M) concentration used. Hence, at the concentrations used in these studies, these agents appear to produce specific inhibition of EGF- and TRH-stimulated rPRL promoter activity without significant indiscriminant effects on transcription of promoter-reporter constructs or translation of reporter enzymes indicating a lack of nonspecific toxic effects. Cells were also examined microscopically after treatment and, with the exception of staurosporine at doses of 10-6 M and higher, no obvious change in cell adhesion or morphology was detected with inhibitor treatment (data not shown).
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In simultaneous experiments, the inhibitors staurosporine, bisindoylmaleimide I, and Calphostin C at 10-6 M completely inhibited phorbol ester (TPA) induced activation of the rPRL promoter. Rottlerin produced a 510% inhibition at 5 µM and complete inhibition at 50-µM concentrations. Go 6976 failed to inhibit TPA-induced promoter activity (data not shown).
Taken together, these data suggest that one or more PKC isozyme(s) is necessary for maximal EGF and TRH stimulation of rPRL transcription. This is supported by the inhibition of both EGF and TRH responses with staurosporine, bisindolylmaleimide I, and Calphostin C. Inhibition by Calphostin C suggests that DAG is a factor in both EGF- and TRH-stimulated rPRL transcription but that different isozymes may be involved in that the TRH effect was more sensitive to inhibition by this agent. It also makes it unlikely that either of the atypical PKC isozymes (PKCs or
) play a substantial role in mediating TRH or EGF effects, as these isozymes are reportedly not DAG dependent in vitro. Data utilizing the selective inhibitor Go 6976 would suggest that one or more of the Ca2+-dependent isozymes (
and/or ßII) may be necessary for maximal EGF response. However, the 10-fold higher than anticipated dose requirement for inhibition by Go 6976 and the lack of complete inhibition at doses more than 100-fold the Ki for this inhibitor would imply that the Ca2+-dependent PKC isozymes are not absolutely required for the EGF response. Lack of inhibition of TRH-activated rPRL promoter activity with Go 6976 suggests that the TRH effect is not dependent upon the Ca2+-dependent PKCs. This finding is surprising in view of the large body of previous studies suggesting an important role for intracellular calcium in mediating TRH effects on PRL gene expression. The effect of Rottlerin suggests that nPKC
may be crucial to both EGF- and TRH-mediated PRL gene expression.
Effect of Stable PKC Overexpression on Basal, TRH-, TPA-, and EGF-Stimulated rPRL Promoter Activity
Previous reports have implicated nPKC as a mediator of both basal and TRH-stimulated PRL secretion (10). nPKC
is the most abundant PKC isozyme found in GH4C1 cells (10, 14, 15). Furthermore, nPKC
appears to be translocated to the plasma membrane and down-regulated in response to TPA and TRH exposure in GH4C1 cells (10, 13, 15, 23); and some studies have suggested temporal correlations between these effects on nPKC
and PRL gene expression (15, 24). Given the close coupling of PRL gene expression and PRL secretion under several physiological and pharmacological conditions, nPKC
would seem a likely candidate to mediate TRH and EGF regulation of the rPRL promoter. The inhibition of TRH effects with Calphostin C but not with Go 6976 also suggested that this member of the novel isozyme family might play a partial role in TRH-mediated PRL transcription. As specific inhibitors of nPKC
have yet to be described, we investigated the effect of nPKC
overexpression on basal, EGF-, TRH-, and TPA-induced rPRL promoter activity.
Two GH4C1 cell lines, E4 and E14, stably overexpressing PKC, were transiently transfected with either the -425 rPRL promoter luciferase reporter construct [or a cfos-81tk (thymidine kinase) luciferase reporter construct] along with the CMVß-galactosidase (gal) construct. Figure 2
depicts the relative rPRL promoter activity in E4 and E14 cells compared with that seen in the GH4C1-derived S1 control cell line and the GH4T2 cell line. No significant difference in rPRL promoter activity was observed with these cell lines in the basal state (Fig. 2A
). All four cell lines also had similar cfos-81tk and CMV promoter activity in the basal state (data not shown).
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Western blot analysis (Fig. 3) of total cell extracts from GH4T2 and from GH4C1-derived S1, E4, and E14 cells clearly demonstrates that nPKC
is overexpressed in the E4 and E14 cells. E4 cells expressed 4.6-fold and E14 cells 7.8-fold higher levels of nPKC
than did the control S1 cell line. The expression of PKC isozymes
, ßII,
, and
were not substantially affected by the overexpression of nPKC
when E4 and E14 cells were compared with S1 cells. The GH4T2 cells did appear to express less of the ßII isozyme than any of the GH4C1 derived cell lines; however, the significance of this is unclear.
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This information is summarized in Table 1. Other than the finding that overexpression of PKC
produced inhibition of both EGF- and TPA-activated rPRL promoter activity, there was no consistent pattern to the effects produced by transient isozyme overexpression based either on the stimulus, the isozyme, or the PKC family. Transient transfections wherein overexpression of PKC isozymes
, ßII,
,
,
, and
was studied utilizing smaller and larger quantities of transfected plasmid (5 and 20 µg) failed to produce more substantial effects on EGF-stimulated rPRL promoter activity (data not shown). In addition, studies were conducted on cells transfected 48 h before extraction and in cells maintained in media containing 10% fetal calf serum after transfection. Although increased isozyme expression could be demonstrated by Western blot analysis and absolute rPRL promoter activity was higher under these conditions, further enhancement of stimulated promoter activity related to specific PKC isozyme expression was not demonstrable (data not shown).
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DISCUSSION |
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Whereas several lines of evidence have previously implicated PKCs in TRH-mediated PRL gene transcription, attempts to clarify the role of PKCs in mediating EGF effects on PRL gene transcription have relied upon the use of phorbol ester exposure to down-regulate PKC levels. Down-regulation is thought to be due primarily to increased proteolysis of membrane-associated (activated) PKCs and in vitro studies have shown that phospholipid activators increase the susceptibility of PKCs to proteolysis (38, 39). Hence, down-regulation has been used both as an indicator of activation and as a tool for determining whether a given response requires PKC. Using this approach, Jackson et al. (28) demonstrated that chronic exposure of GH3 cells to TPA (324 nM for 2 d) inhibited subsequent acute TPA stimulation of rPRL promoter activity but did not inhibit EGF stimulation (measured 2048 h later). Acute stimulatory effects of EGF and TPA on expression of the PRL gene appeared to be additive. These data were taken to imply that EGF and TPA effects were mediated via different mechanisms and hence cast doubt on the role of PKCs in the EGF response. Since that time, however, the complexity of PKC isozyme expression and regulation has been further elucidated. Although phorbol ester (TPA) can produce down-regulation of all except the atypical PKCs, there are different susceptibilities. Specifically, in GH4C1 cells, cPKCßII and nPKC have been reported to be more susceptible to TPA down-regulation than are cPKC
and nPKC
(10, 13, 14). It is unclear from the studies of Jackson et al. (28) to what extent specific PKC isozymes were depleted by the conditions used and, hence, despite the fact that additional TPA failed to further stimulate PRL gene transcription, these data remain inconclusive. Our studies in GH4T2 cells suggest a relative susceptibility of inhibition of ßII >
=
>
at 100200 nM TPA for 24 h. Furthermore, we have been unable to produce more than a 7080% reduction in PKC
protein expression in GH4T2 cells with TPA at 500 nM for up to 48 h (our unpublished data). If activation of only a small proportion of the necessary PKC isozyme(s) is sufficient to convey the EGF signal to the rPRL promoter, pretreatment with TPA may be inadequate to inhibit this response.
Translocation of PKC has also been used as an indication of enzyme activation. Early studies indicated that in many cell types treatment with phorbol esters or growth factors/hormones promoted translocation of PKCs from soluble cell fractions to insoluble crude membrane fractions and that this was associated with enzyme activation. More recently, it has been demonstrated that PKCs undergo intracellular trafficking in response to various stimuli involving the cytoplasm, cytoskeleton, plasma membrane and nucleus and that this translocation likely involves specific high affinity PKC binding proteins in addition to interactions with phospholipids (8, 9). It has thus become clear that down-regulation and translocation are not interchangeable measures of in vivo isozyme-specific PKC function and enzyme activation. In GH4C1 cells, TRH appears to only down-regulate expression of nPKC, and not cPKC
, cPKCßII, nPKC
, or aPKC
(13, 14, 15), whereas, as indicated above, TPA can down-regulate all of these isozymes except aPKC
. Yet both TRH and TPA induce translocation of isozymes
, ßII,
,
, and
from the cytosolic fraction to a crude membrane fraction (13, 14, 15). Immunofluorescence microscopy has demonstrated translocation of PKC
specifically to the plasma membrane in response to TRH and TPA (10). Specific subcellular translocation of other PKC isozymes in GH3, GH4T2, or GH4C1 cells has not been well studied.
Studies on the effect of EGF in down-regulating PKC isozymes in GH4 cells have not been reported although our preliminary studies suggest neither PKC, ßII,
, or
are significantly down-regulated in response to 2550 nM EGF for 24 h (Pickett, C. A., and N. Manning, unpublished data). Translocation of PKC isozymes in response to EGF has not been well characterized in GH3, GH4, or GH4C1 somatolactotrophs. Studies in other cell lines suggest that there are tissue-specific differences in translocation of PKC isozymes in response to EGF as has been observed with other cell stimuli (40, 41, 42, 43, 44). Further studies of translocation, down-regulation, and phosphorylation will likely be useful in elucidating the role of specific PKC isozymes in mediating EGF effects.
Our studies suggest several differences between TRH- and EGF-mediated activation of the rPRL promoter. One striking difference was observed with the inhibitor Go 6976, an agent that appears to be fairly specific in vitro for the Ca2+-dependent PKC isozymes. TRH activation of the rPRL promoter was not inhibited even at the highest concentration of this agent, whereas EGF effects were inhibited at 100 nM concentrations. These findings suggest that the previously well-documented Ca2+ dependence for TRH activation of the rPRL promoter and the synergy observed between Ca2+ and both EGF and TRH responses (18) is likely a result of other Ca2+-dependent processes and not directly via an effect on one or more of the Ca2+-dependent isozymes of PKC. Previous studies by Bandyopadhyay and Bancroft (45) have also indicated that the enhanced expression of PRL mRNA observed with increased intracellular calcium in GH3 cells is PKC independent. Other differences include the relative sensitivity to Calphostin C and the mild activation of EGF but not TRH response produced by overexpression of nPKC.
The inhibition of both EGF- and TRH-induced rPRL promoter activity by Rottlerin, at concentrations as low as 1 µM, provides support for a role of PKC in mediating regulation of PRL gene expression. Recent studies have demonstrated that fibroblast growth factor (FGF) activation of the rPRL promoter in GH4 cells is dependent upon PKC
(46). In these studies both FGF2 and FGF4 induced rPRL promoter activity were inhibited by both Caphostin C and Rottlerin and this response blocked by adenoviral dominant negative PKC
expression. PKC
has also recently been implicated in EGF-induced expression of the cyclin-dependent kinase inhibitor p21 in A431 cells (47) as well as in the regulation of several other TPA-inducible genes (48, 49).
Some controversy still exists as to the specificity of Rottlerin in inhibiting PKC. In addition to PKC
, Rottlerin does inhibit the calmodulin-dependent kinase-III (or elongation factor-2 kinase) at concentrations of 5 µM (36, 37). One recent study has questioned the specificity of Rottlerin based on in vitro studies in which concentrations of 100 µM Rottlerin were required to inhibit PKC
, whereas inhibition of MAPKAP-K2, p38 regulated/activated protein kinase, and PKA was observed at concentrations of 20 µM (50). In contrast, several studies utilizing overexpression of PKC
have demonstrated inhibition at 0.15.0 µM Rottlerin of PKC
-dependent effects in vivo (48, 51, 52) and with purified PKC
in vitro (36, 53). In our studies, bisindolylmaleimide I also significantly inhibited the EGF and TRH effects, whereas this agent does not appear to inhibit MAPKAP-K2, p38 regulated/activated protein kinase, or PKA in vitro (50). Furthermore, calmodulin-dependent kinase-III/elongation factor-2 kinase is resistant to inhibition by staurosporine with an IC50 of more than 50 µM for staurosporine (37), whereas EGF- and TRH-stimulated rPRL promoter activation was inhibited at nanomolar concentrations. Hence, it seems unlikely that any of these kinases are responsible for the EGF and TRH responses observed in our studies.
Given the evidence for PKC involvement in EGF- and TRH-mediated rPRL promoter activity provided by the inhibitor data, and particularly the evidence for a specific role of PKC, we had anticipated that overexpression of PKC
and potentially several other PKC isozymes would enhance these responses. However, only nPKC
produced statistically significant EGF-enhanced activity. Western blot analysis clearly revealed enhanced expression with transient transfection of PKCs
, ßII,
,
,
, and
and in cells stably overexpressing PKC
. Similar studies have demonstrated enhanced basal and TRH induced PRL secretion in GH4C1 cells stably and transiently transfected with PKC
(10). Furthermore, overexpression of several PKC isozymes has been shown to lead to enhanced expression of specific genes in other cell lines (54). One would anticipate that the success of such experiments would be dependent upon the level of endogenous expression of a given isozyme(s), the relative fraction of activated enzymes required to produce a given response, and the limitation of downstream effectors. The lack of enhancement of not only EGF- and TRH- but of TPA-mediated rPRL promoter activity by overexpression of PKC isozymes in our studies is highly suggestive that a small fraction of activated kinase(s) can produce a full response or that a downstream effector is limiting.
Previous studies have demonstrated that overproduction of native nPKC enhanced both basal and TRH-stimulated PRL secretion. Overexpression of other endogenous PKC isozymes
, ßII, and
did not affect secretory responses. A modest 2- to 4-fold increase in nPKC
seemed to confer a significant increase in TRH-stimulated PRL secretion from transfected GH4C1 cells, whereas higher levels of nPKC
expression decreased secretion to control levels (13). Thus, a constitutively high activity of nPKC
may result in feedback inhibition of the secretory system. Several other studies have demonstrated that marked overexpression of exogenous cPKCs can result in inhibition of various PKC associated cellular responses (55, 56, 57). In our transient transfection studies, we did examine basal and EGF-stimulated rPRL promoter activity in cells transfected with varying amounts (520 µg) of the wild-type PKC isozyme constructs without observing evidence of biphasic responses. Although unlikely, we cannot entirely exclude the possibility that the lack of enhancement of stimulated rPRL promoter activity in our studies of overexpression was the result of some type of feedback inhibition.
Interestingly, overexpression of nPKC, which like nPKC
is a member of the novel PKC isozyme family, did produce an increase in EGF stimulated rPRL promoter activity. Previous studies have suggested that in GH4/GH4C1 cells, under typically employed cell culture conditions, nPKC
is expressed only at very low levels (10, 46). As the IC50 of nPKC
for Rottlerin has been reported to be 82 µM (36), it would seem doubtful that this isozyme normally conveys the EGF response in GH4/GH4C1 cells. Whether this isozyme can substitute for nPKC
when overexpressed remains to be clarified.
In summary, our studies indicate that EGF- and TRH-stimulated rPRL promoter activation in GH4T2/GH4C1 cells are both mediated by several members of the PKC family. Differences in susceptibility to selective PKC inhibitors and to overexpression of PKC isozymes suggest divergence in the relative role of specific isozymes in conveying EGF and TRH signals. The novel PKC isozyme, nPKC, appears to be the most crucial in PRL transcriptional regulation rather than the nPKC
isozyme, which has been implicated in TRH regulation of PRL secretion. These studies are compatible with previous TPA down-regulation studies in that cPKC
, novel and atypical PKC isozymes appear to be less sensitive to down-regulation. The finding that overexpression of wild-type isozymes failed to substantially enhance the EGF or TRH responses, and that kinase negative constructs produced little or no inhibition, suggests that only a relatively small proportion of activated kinase(s) is necessary to mediate these responses or that the mediator(s) downstream of PKC is (are) limiting in regards to PRL promoter activation. Because multiple isozymes appear to be involved in both responses, compensation by one isozyme when another is inhibited may also occur. Further studies will be necessary to determine the mechanism by which PKC activation mediates PRL gene transcription.
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MATERIALS AND METHODS |
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Plasmids and Transfections
All transient transfections were performed with 5 µg of the rPRL promoter luciferase (pA3rPRLluc) reporter construct, as previously described (20, 58, 59). The pA3rPRLluc construct contains the firefly luciferase coding region under the control of a 498-bp fragment (-425 to +73) of the proximal rPRL promoter downstream of three polyadenylation termination sites in pA3 luc. In all electroporations, 300 ng of a pCMVß-gal plasmid or 5 µg of a pSVß-gal plasmid was included as an internal control for transfection efficiency and as an indicator of the specificity of the EGF, TRH, TPA, and effector DNA responses (20, 58, 59). The following plasmids encoding intact PKC isozymes under control of the SV40 promoter in the SRD vector (10, 60, 61) were employed: SRD PKC (YK529), encoding the -196 to 2554 region of rabbit nPKC
(10, 61), SRD
(YK504), encoding the -4 to 2648 region of rabbit cPKC
(62), SRDßI (M136) encoding the -126 to 2092 region of rabbit cPKCßI (62), SRDßII (M130) encoding the -126 to 2972 region of rabbit cPKCßII (62), SRD
(M241) encoding the -13 to 2525 region of mouse nPKC
(63), SRD
encoding the -55 to 2117 region of mouse nPKC
(64), SRD
(MLNP45) encoding the full length cDNA of mouse aPKC
(65), SRD
(M129) encoding the -31 to 2301 region of rabbit cPKC
(54, 61), SRD
encoding the -23 to 2262 region of mouse nPKC
(63, 66), and SRD
(M246) encoding the -3 to 2160 region of mouse aPKC
(63). Briefly, these cDNA expression vectors were constructed by introducing a cDNA fragment encoding the PKC between the PstI and KpnI sites of the pcDL-SR
296 vector, which was a derivative of the Okayama-Berg expression vector pcD (60, 67). The SRD control plasmid (YK539) contains an additional EcoRI linker (60). In addition, PKC isozyme kinase inactive constructs, wherein Lys has been substituted for Arg in the ATP binding domain, were also used and include: PKC
KmR, PKCßIIKmR, PKC
KmR, PKC
KmR, and PKC
KmR (10, 68). The total amount of DNA was kept constant using the empty SRD vector (YK539) or pGEM7 (Promega Corp., Madison, WI) DNA. All plasmid DNAs were purified according to the QIAGEN Mega protocol (QIAGEN Inc., Chatsworth, CA) and quantified as described previously (69).
Transient transfections were performed in triplicate by electroporation of aliquots containing approximately 2 x 106 cells added to plasmid DNA. After electroporation, cells were plated in 3 ml of DMEM with reduced fetal calf serum (0.6%) with G418 where appropriate, and incubated for 2448 h post transfection. EGF was added at a final concentration of 25 nM to the existing medium for the last 6 h of incubation. TRH and TPA [solubilized in dimethylsulfoxide (DMSO)] were added at 100 nM concentration for the last 6 h of the incubation. Cells were harvested and reporter enzyme activities determined as described previously (20, 26, 27).
Analysis of Data
PRL promoter activity was determined as relative light units (total light units produced by luciferase normalized to total ß-gal activity). Data are presented as relative promoter activity, which is equivalent to EGF, TPA, or TRH fold activation over basal promoter activity in untreated cells. In Fig. 1, CMV or SV ß-gal activity is also shown relative to expression in basal untreated cells. Data are the mean of three to seven separate experiments ± SEM unless otherwise specified. For statistical analysis, P values were calculated by Students t test. Additionally, several preparations of a given plasmid construct were used over the course of these studies.
Western Blot Analysis
For Western blot analysis cells were extracted in ice-cold RIPA buffer (PBS, 1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% sodium dodecyl sulfate) containing 100 mM phenylmethylsulfonyl fluoride and protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Molecular Biochemicals, Indianapolis, IN). Viscosity was reduced by shearing the extract through a 23-gauge needle and insoluble material was removed by centrifugation. Protein content of the extracts was measured using the BCA assay (Pierce Biochemicals, Rockford, IL) and 80100 µg protein subjected to sodium dodecyl sulfate denaturing gel electrophoresis on 10% polyacrylamide. After transfer to nitrocellulose, blots were probed with a 1:500 dilution of isozyme-specific rabbit anti-PKC antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and labeled with antirabbit horseradish peroxidase (enhanced chemiluminescence, Amersham Pharmacia Biotech). Labeled protein was quantitated using a Molecular Dynamics (Sunnyvale, CA) Laser-scanning densitometer with ImageQuant software.
PKC Inhibitors
All PKC inhibitors were purchased from Calbiochem (La Jolla, CA). Staurosporine, bisindolylmaleimide I (Go 6850, GF109203X), Go 6976, and Calphostin C (UCN 1028c) were solubilized in DMSO before addition to cell media. Rottlerin was solubilized in either 100% ethanol or DMSO. Inhibitors were added 20 min before addition of EGF, TRH, or TPA. In experiments using Calphostin C, cells were exposed to light both before the addition of EGF, TRH, or TPA and throughout the subsequent 6-h incubation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Current address for Y.A.: Department of Laboratory Animal Science, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
Abbreviations: aPKC, Atypical PKC; CMV, cytomegalovirus; cPKC, Ca2+-dependent PKC; DAG, diacylglycerol; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; FGF, fibroblast growth factor; gal, galactosidase; Ki, inhibitory constant; nPKC, novel or Ca2+-independent PKC; PKC, protein kinase C; PRL, prolactin; rPRL, rat PRL; PS, phosphatidyl-L-serine; SV, Simian virus; tk, thymidine kinase; TPA, 12-O-tetradecanoyl phorbol-13-ester.
Received for publication November 12, 2001. Accepted for publication September 12, 2002.
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REFERENCES |
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