INTRODUCTION

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THE MITOCHONDRIAL AND cytosolic isoforms of phosphoenolpyruvate carboxykinase (PCK) are expressed primarily in liver, kidney, and adipose tissue, where they catalyze the rate-limiting steps in gluconeogenesis and glyceroneogenesis (12). In liver, gluconeogenesis functions to sustain normal blood glucose levels during a fasted state, whereas in kidney, this process is associated with the catabolism of glutamine and is coupled to ammoniagenesis and the maintenance of acid-base balance. In adipose tissue, glyceroneogenesis participates in the synthesis and storage of triglycerides. Thus the PCK activity must be differentially regulated in a manner consistent with its tissue-specific function. However, unlike many other key regulatory enzymes, this activity is neither affected by allosteric modulators nor subject to covalent modification (12). Furthermore, expression of the mitochondrial isozyme appears to be constitutive. Only the expression of the single-copy gene that encodes the cytosolic isozyme is subject to tissue-specific regulation by multiple hormones and effectors (12). For example, transcription of this gene in liver is enhanced by glucocorticoids, glucagon (via cAMP), and thyroid hormone, whereas insulin and activation of protein kinase C by phorbol esters act as dominant negative regulators. In contrast, expression in kidney is increased by cAMP, glucocorticoids, and metabolic acidosis and is decreased during metabolic alkalosis. Finally, expression in adipocytes is initiated during differentiation and development and is inhibited by glucocorticoids (12).

This complex pattern of regulation is achieved primarily through an equally complex proximal promoter which accounts for much of the developmental, tissue-specific, and metabolic regulation of the transcription of the cytosolic PCK gene (19, 24). The promoter segment extending to 490 bp from the transcription initiation site contains at least 13 distinct protein binding sites (12). Tissue-specific regulation is achieved by the differential expression of specific transcription factors that utilize different combinations of the promoter elements. For example, cAMP-dependent regulation of PCK gene expression in HepG2 liver cells is achieved through the binding of the cAMP response element binding protein (CREB) to the CRE-1 element (26) and of a CAATT/enhancer binding protein (C/EBP) and c-jun to an upstream segment consisting of the P3(I), P3(II), and P4 elements (25, 27). In contrast, cAMP-dependent regulation in LLC-PK₁-F⁺ renal tubular epithelial cells requires only the CRE-1 and the P3(II) elements (18). The dominant negative regulators may also utilize the same elements that mediate the stimulatory effects of enhancers. For example, the inhibitory effects of insulin are mediated through an element that constitutes part of the glucocorticoid regulatory unit (22) and potentially through CRE-1 (23).

Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also causes a rapid inhibition of transcription of the PCK gene in liver cells (3). Like insulin action, this response reverses the stimulatory effects of glucocorticoids and cAMP. The effect of PMA also maps, at least in part, to the insulin response element that is located within the glucocorticoid regulatory unit (21). However, the effects of insulin and PMA are additive (4) and are responsive to different kinase inhibitors (31), suggesting that they utilize different signal transduction pathways. In the current study, the effects of PMA and staurosporine on PCK gene expression were studied using LLC-PK₁-F⁺ kidney cells. Activation of protein kinase C by PMA caused a rapid decrease in PCK mRNA levels that was dominant over the stimulatory effects of cAMP and the exposure to acidic medium. Furthermore, downregulation of protein kinase C by long-term incubation with PMA resulted in increased levels of PCK mRNA. Although the negative effect of PMA was reversed by staurosporine, the two antagonists were shown to act primarily through separate effects on PCK mRNA stability and transcription, respectively.

	MATERIALS AND METHODS

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Materials

Methods

Northern analysis. Total RNA was isolated from cultured cells using the acid guanidinium thiocyanate method (2). Electrophoresis, transfer to GeneScreen Plus, hybridization, and washing of blots were carried out as described previously (14). The probes used for hybridization included the rat cytosolic PCK cDNA (32) and the porcine urokinase plasminogen activator (uPA) cDNA (5). The blots were stripped of the initial probe and rehybridized with a -actin cDNA (9). All probes were labeled with [-³²P]dCTP using the Pharmacia oligolabeling kit. Quantification of mRNA levels was accomplished using a Molecular Dynamics PhosphorImager. In all experiments, the levels of PCK and uPA mRNAs were standardized relative to those of -actin mRNA. For the half-life determinations, transcription was blocked by adding 65 µM 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) as described previously (11).

Cell fractionation. Confluent cultures of LLC-PK₁-F⁺ cells were treated with 0.4 µM PMA and harvested by scraping the cells in 10 ml of extraction buffer (150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 50 mM Tris-Cl, pH 7.5). The cells were pelleted by centrifugation at 300 g for 10 min and then resuspended in 1 ml of extraction buffer containing 25 µg/ml leupeptin and 250 mU/ml aprotinin. The cell suspensions were lysed by repeated (3×) freezing in liquid nitrogen and thawing at 37°C. The crude cell lysates were centrifuged at 17,000 g for 15 min at 4°C. The supernatants were then centrifuged at 430,000 g for 10 min at 4°C. The resulting supernatants were the cytosolic fractions. The pellets from the two centrifugation steps were combined and incubated in 0.2 ml of extraction buffer containing 1% Triton X-100 for 20 min to solubilize the membrane-bound protein kinase C. The samples were then centrifuged for 10 min at 430,000 g, and the resulting supernatants were the solubilized membrane fractions.

Western blotting. Samples of the cytosolic and solubilized membrane fractions containing 15 µg of protein were separated by SDS-PAGE using a 10% polyacrylamide slab gel. The separated proteins were electrophoretically transferred to an Immobilon-P polyvinylidene fluoride microporous membrane and then incubated with a rabbit polyclonal antibody specific for the -isozyme of protein kinase C (PKC). The resulting complex was further conjugated with a horseradish peroxidase-linked anti-rabbit IgG antibody and visualized using the Amersham ECL system and Hyperfilm-ECL.

Chloramphenicol acetyltransferase assays. The various block mutations (17) of the chloramphenicol acetyltransferase (CAT) gene driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK₁-F⁺ cells (PCK₄₉₀CAT) were obtained from Richard Hanson (Case Western Reserve University). LLC-PK₁-F⁺ cells were split and replated at 30% confluence. The cells were grown for 1-12 days in culture and then transfected by calcium phosphate precipitation of DNA (11). The DNA samples contained 10-15 µg of the PCK-CAT construct and 2-5 µg of pRSVgal. With confluent cultures, the excess precipitate was removed after 8 h, and the cells were washed with phosphate-buffered saline and refed for 16 h with normal medium. In contrast, subconfluent cultures were incubated with the precipitated DNA for the full 24 h. The medium was then replaced with normal medium containing the indicated supplements. After 16-20 h, the cells were homogenized and assayed for -galactosidase activity. Samples of homogenate (50-100 µl) containing equivalent units of -galactosidase activity were used to measure CAT activity (15). The acetylated products and the unreacted substrate were separated by thin-layer chromatography, and the percent conversion was quantitated using a PhosphorImager.

	RESULTS

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Effect of PMA

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of phorbol 12-myristate 13-acetate (PMA) on the level of phosphoenolpyruvate carboxykinase (PCK) mRNA in LLC-PK1-F+. Cells were initially grown for 12 days in normal (pH 7.4, 25 mM HCO3) medium. They were then incubated for 18 h in fresh normal medium (pH 7.4), acidic (pH 6.9, 10 mM HCO3) medium, or normal medium containing 0.5 mM 8-(4-chlorophenylthio)-cAMP (+cAMP) and then treated for 4 h in the absence () or presence (+) of 1 µM PMA (A). Alternatively, the cells were maintained in normal (pH 7.4) or acidic (pH 6.9) medium for 24 h in the absence () or presence (+) of 0.4 µM PMA (B). Samples containing 15 µg of total RNA were fractionated by electrophoresis, transferred to GeneScreen Plus, and probed for PCK mRNA. Relative intensities of the resulting bands were quantitated with a PhosphorImager. Each bar represents the mean ± SE of 6-8 RNA samples isolated from separate plates of cells.

The addition of PMA causes a rapid activation and a gradual downregulation of the PKC. This effect was quantitated by Western blot analysis of cytosolic and membrane fractions isolated from LLC-PK₁-F⁺ cells (Fig. 2). In unstimulated cells, the PKC protein was detected only in the cytosolic fraction. However, within 5 min after addition of 0.4 µM PMA, all of the PKC protein was translocated to the membrane fraction. Membrane association, which is an essential step in the activation of PKC, was sustained by the continued incubation of the LLC-PK₁-F⁺ cells with PMA. Furthermore, the level of the active PKC protein remains constant for up to 1 h after addition of PMA. However, after 2-4 h of incubation, the level of membrane-associated PKC decreased slightly, and by 24 h no PKC protein could be detected in either the cytosolic or membrane fractions.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of the effect of PMA on the translocation and downregulation of -isozyme of protein kinase C (PKC) in LLC-PK1-F+ cells. Confluent cultures of LLC-PK1-F+ cells were incubated with 0.4 µM PMA for the indicated periods of time. Samples containing 15 µg of protein from the cytosol (top) and membrane (bottom) fractions were separated by SDS-PAGE, immunoblotted, and stained with an anti-PKC antibody. A rat brain cytosolic extract was run as a positive control (right lanes). All of the immunostaining bands comigrated as an 80-kDa protein. Data are representative of 3 separate experiments.

The effect of PMA was blocked by the addition of specific inhibitors of protein kinase C (Fig. 3). Treatment of LLC-PK₁-F⁺ cells for 4.5 h with either 1 µM Gö-7874 or 2.5 µM Ro-31-8220 caused a slight (1.5-fold) increase in the levels of PCK mRNA. In contrast, treatment for 4 h with 1 µM PMA caused a dramatic decrease in PCK mRNA levels. However, when the cells were pretreated with either inhibitor for 30 min before and then during the incubation with PMA, the levels of the PCK mRNA were decreased to only 50-70% of that observed in untreated cells. The substantial protection afforded by the specific inhibitors strongly suggests that the effect of PMA is mediated by activation of a protein kinase C.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Role of protein kinase C in the PMA-induced decrease in PCK mRNA levels. Cells were initially grown for 12 days in normal (pH 7.4, 25 mM HCO3) medium. They were then incubated for 30 min in fresh medium in absence or presence of either 1 µM Gö-7874 or 2.5 µM Ro-31-8220. Cells were then incubated for 4 h in absence () or presence (+) of 1 µM PMA. Samples containing 15 µg of total RNA were fractionated by electrophoresis, transferred to GeneScreen Plus, and probed for PCK mRNA. Relative intensities of the resulting bands were quantitated with a PhosphorImager. Each bar represents the mean ± SE of 3 RNA samples isolated from separate plates of cells.

The kinetics of the PMA effect are rapid (Fig. 4). When LLC-PK₁-F⁺ cells were maintained in acidic medium for 16 h and then treated with PMA, the resulting decrease in PCK mRNA was initiated after a 30-min lag, proceeded with first-order kinetics, and had an apparent half-life of 1 h. In contrast, the decrease in PCK mRNA levels caused by simply transferring the cells from acidic to normal medium also exhibited a first-order decay but had an apparent half-life of 4 h. The latter value is similar to that previously measured (15) when cells grown in either normal or acidic medium were treated with DRB, a specific inhibitor of RNA polymerase II (6). Thus the rapid and pronounced decrease caused by addition of PMA is due, at least in part, to a stimulation of the rate of degradation of the PCK mRNA. This effect is specific, since the level of the stable -actin mRNA remains constant for 4 h following treatment with PMA or following the transfer from acidic to normal medium.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Comparison of effects of PMA and of recovery from acidic medium on the rate of decrease in PCK mRNA in LLC-PK1-F+ cells. Cells were initially grown in normal medium for 10 days and then incubated for 16 h in acidic medium. One set of adapted cells was then treated with 1 µM PMA in acidic medium while the second was returned to normal medium. Total RNA samples were isolated either immediately before or at various times either after adding PMA (, pH 6.9 + PMA) or after the start of recovery (, pH 6.9  pH 7.4). Samples containing 15 µg of total RNA were fractionated by electrophoresis, transferred to GeneScreen Plus, and probed for PCK mRNA. Relative intensities of the resulting bands were quantitated with a PhosphorImager. Each point represents the mean of duplicate samples in which the variance was less than 20% of the mean. Reported data are representative of 2 experiments.

Effect of Staurosporine

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of PMA and of staurosporine on the levels of PCK and urokinase plasminogen activator (uPA) mRNAs in LLC-PK1-F+ cells. Cells were initially grown in normal medium for 10 days and then incubated in fresh normal medium for 16 h. Then they were treated for 6 h in absence () or presence (+) of 1 µM PMA and/or the indicated amounts of staurosporine (Stauro). Samples containing 15 µg of total RNA were fractionated by electrophoresis, transferred to GeneScreen Plus, and probed for PCK (A), -actin (B), and uPA mRNAs (C). Relative intensities of the resulting bands were quantitated with a PhosphorImager. Data are means ± SD of 3 RNA samples isolated from separate plates of cells.

The addition of staurosporine to LLC-PK₁-F⁺ cells has no effect on the apparent half-life of the PCK mRNA (Fig. 6). Confluent cultures of LLC-PK₁-F⁺ cells were treated in the presence or absence of 250 nM staurosporine for 12 h, and then 65 µM DRB was added to inhibit transcription. Pretreatment with the higher level of staurosporine was used to produce a 10-fold increase in the level of PCK mRNA. After addition of DRB, the levels of PCK mRNA in the two sets of cultures decreased with similar kinetics. The apparent half-lives measured in the presence and absence of staurosporine were 3.5 and 4 h, respectively. This result provides further evidence that staurosporine was not acting solely to reverse the PMA-stimulated degradation of the PCK mRNA.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of staurosporine on the apparent half-life of PCK mRNA in LLC-PK1-F+ cells. Cells were grown for 12 days in normal medium and then incubated in normal medium for 12 h in absence (control) or presence (+staurosporine) of 250 nM staurosporine. Total RNA was isolated either immediately before or at various times after adding 65 µM 5,6-dichloro-1--D-ribofuranosylbenzimidazole. Samples containing 15 µg of total RNA were fractionated by electrophoresis, transferred to GeneScreen Plus, and probed for PCK mRNA. Relative intensities of the resulting bands were quantitated with a PhosphorImager. Each point represents the mean of duplicate samples in which the variance was less than 25% of the mean. Data were normalized to the mean value of the zero time point obtained in the absence of staurosporine.

The ability of staurosporine to stimulate transcription of the PCK gene was tested by determining its effect on the CAT activity measured in subconfluent cultures of LLC-PK₁-F⁺ cells transiently transfected with PCK₄₉₀CAT (Fig. 7). This construct contains the entire proximal promoter of the PCK gene. The addition of 25 nM staurosporine caused a ninefold increase in CAT activity. The observed stimulation is similar to that caused by the addition of cAMP. However, the stimulatory effects of cAMP and staurosporine were additive. These results suggest that staurosporine acts to stimulate transcription, possibly through inhibition of a protein kinase activity, which normally suppresses transcription of the PCK gene in LLC-PK₁-F⁺ cells.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Comparison of the effect of staurosporine, PMA, and cAMP on the chloramphenicol acetyltransferase activity in subconfluent cultures of LLC-PK1-F+ cells. Cells were grown for 2 days and then transfected with PCK490CAT. After 1 day, cells were pretreated for 30 min in absence () or presence (+) of 25 nM staurosporine (Staur) and/or 1 µM PMA. Cells were then incubated for 20 h with or without the addition of 0.5 mM CPT-cAMP. Cells were then homogenized and assayed for CAT activity. Bottom spots on the chromatogram are the unreacted [14C]chloramphenicol, and top 2 rows of spots are the acetylated products. Chromatogram was imaged and quantitated on a PhosphorImager. Numbers at the bottom are the mean of the percent conversion (% conv) of starting material to product for duplicate transfections. Data are representative of 3 separate experiments.

The measurement of PCK₄₉₀CAT activity was also used to determine whether the addition of PMA could affect transcription of the PCK gene. The addition of PMA failed to reverse the stimulatory effect of staurosporine or the additive effects of staurosporine and cAMP (Fig. 7). Furthermore, the addition of PMA had no effect on the basal or cAMP-stimulated PCK₄₉₀CAT activity measured in LLC-PK₁-F⁺ cells that were grown (1-12 days) to different stages of confluence and differentiation (data not shown). Similarly, PMA had no effect on the expression of a larger CAT construct that contained the initial 2 kb of the PCK promoter. Thus PMA activation of protein kinase C has no effect on transcription from the proximal promoter of the PCK gene in LLC-PK₁-F⁺ cells.

Various block mutations of the PCK₄₉₀CAT construct were used to map the promoter elements (Fig. 8) that mediate the stimulatory effect of staurosporine. Each of these constructs contains the entire 490 to +73 bp segment of the PCK gene but contains a substitution of 5-15 bp in one of the identified promoter elements (17). As reported previously (18), all of the constructs except for the P2 and CRE-2 block mutations exhibit similar levels of basal activity when transfected into subconfluent cultures of LLC-PK₁-F⁺ cells (Fig. 9). Mutations of the P1 and P3(I) elements had little effect on the ability of staurosporine to stimulate the CAT activity. The mutation of the CRE-2, P2, or P4 elements reduced the stimulation by staurosporine to ~50% of that observed in the wild-type construct. However, mutation of the CRE-1 or P3(II) elements reduced the stimulation to ~17%. The CRE-1 and P3(II) elements contain 8- and 7-bp sequences that match the consensus for CREB and AP-1 binding sites, respectively (Fig. 8). As indicated, five of the consensus base pairs were mutated to create the corresponding block mutations of the two elements. Thus the same two elements that mediate the pH-responsive (15) and the cAMP-dependent (18) induction of PCK gene in LLC-PK₁-F⁺ cells are also primarily responsible for mediating the effect of staurosporine.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Proximal promoter region of the rat cytosolic PCK gene. Locations of the cis-acting elements of the PCK promoter are drawn to scale. Sequences of the CRE-1 and P3(II) elements and of their corresponding block mutations are also illustrated.

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Mapping of the promoter elements that mediate the effect of staurosporine on the activity of PCK490CAT transfected into LLC-PK1-F+ cells. Cells were grown for 1 day and then transfected with the various block mutations of the PCK490CAT construct. After 1 day, cells were treated for 16 h in absence (solid bars) or presence of 25 nM staurosporine (hatched bars) and then assayed for CAT activity. Data are the means ± SD of 3 separate transfections. Numbers above the bars indicate the increase in CAT activity measured in the presence of staurosporine vs. those measured in the corresponding control. The specific elements within the PCK promoter that were mutated are indicated below the bars. The 490 construct contains the nonmutated promoter.

	DISCUSSION

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The mechanisms of the various effectors that alter the levels of PCK mRNA in LLC-PK₁-F⁺ cells are summarized in Table 1. Acidic pH, cAMP, and staurosporine are all positive effectors which stimulate transcription of the PCK gene. However, the observed responses occur with different kinetics. Furthermore, the effects of cAMP and of staurosporine are additive. Thus the three effectors may act through separate signal transduction pathways. In contrast, PMA is a negative effector that acts primarily by inducing a more rapid degradation of the PCK mRNA. Because of its magnitude and unique mechanism, the negative effect of PMA is dominant over the 2.5- to 3-fold stimulation of transcription caused by addition of acidic medium or cAMP to confluent cultures of LLC-PK₁-F⁺ cells.

The current study further illustrates the differential regulation of PCK gene expression in liver and in kidney. In hepatoma cells, the addition of PMA leads to a rapid and pronounced inhibition of transcription (3). This effect maps to an insulin response element (21) but proceeds through a separate signaling pathway (31) and is additive to the effects of insulin (4). In LLC-PK₁-F⁺ kidney cells, the addition of PMA produces an equally rapid and pronounced effect on the levels of PCK mRNA. However, this response is not due to an inhibition of transcription that is mediated by elements within the proximal promoter but instead is achieved, at least in part, through an increased rate of degradation of the PCK mRNA. Both cellular responses appear to be mediated through activation of protein kinase C. In both systems, the response is initiated only by phorbol esters, which are known activators of protein kinase C, and the response is blocked when protein kinase C is downregulated following prolonged exposure to a high concentration of PMA. In liver cells, the effect is also reproduced by addition of 1,2-dioctanoylglycerol. In the LLC-PK₁-F⁺ cells, the addition of repeated doses of 1,2-dioctanoylglycerol caused only a slight reduction of PCK mRNA levels (data not shown). Thus the kidney cells may more rapidly degrade diacylglycerides.

In hepatoma cells, the PCK mRNA has an apparent half-life of ~30 min (12). Thus inhibition of transcription produces a rapid decrease in the level of the PCK mRNA. However, the apparent half-life of the PCK mRNA in LLC-PK₁-F⁺ cells is ~4 h. This was previously demonstrated by using DRB to inhibit transcription (15). In the current study, a similar half-life was measured when the rate of transcription was rapidly decreased by returning pH 6.9-adapted cells to normal medium. This observation confirms the validity of using DRB as a selective transcriptional inhibitor to measure mRNA half-lives. Given the greater stability of the PCK mRNA in the LLC-PK₁-F⁺ cells, a PMA-mediated inhibition of transcription would have produced a more gradual decrease in PCK mRNA levels. However, by stimulating PCK mRNA degradation, the resulting effect of PMA addition occurs with similar kinetics in the two cell lines.

The parent LLC-PK₁ cells express significant levels of only the - and -isoforms of protein kinase C (1). Treatment of these cells with PMA results in the membrane translocation of both isoforms but only the PKC protein is downregulated by prolonged exposure to PMA. However, after 24 h of treatment with 0.4 µM PMA, PKC was still detectable, although at very low levels (E. Hütter, N. Spitaler, E. Feifel, and G. Gstraunthaler, unpublished observations). Chronic treatment of LLC-PK₁ cells with PMA also leads to the reduced expression of various proximal tubule-specific properties (1). In contrast, treatment of LLC-PK₁-F⁺ cells, a gluconeogenic substrain of LLC-PK₁ cells, with 0.4 µM PMA produced a more rapid membrane translocation and the complete downregulation of PKC. The effects of PMA on PKC activity correlated very well with the observed changes in PCK mRNA. Furthermore, pretreatment of the LLC-PK₁-F⁺ cells with either of two specific inhibitors of protein kinase C greatly reduced the acute effect of PMA. These findings further support the conclusion that the observed effects of PMA on the levels of the PCK mRNA are mediated by protein kinase C.

The cumulative data suggest that protein kinase C is largely inactive in confluent and well-differentiated cultures of LLC-PK₁-F⁺ cells. Thus various growth factors or mitogenic peptides that are physiological activators of protein kinase C within the renal proximal tubule may produce similar effects. Activation of this signaling pathway may play an important role in preparing cells to undergo dedifferentiation and eventual cell division. The loss of PCK activity, as well as the associated capacity to carry out gluconeogenesis, would be essential to produce glycolytic cells that grow and replicate rapidly.

The 3'-nontranslated region of the PCK mRNA contains specific protein binding sites (20) that mediate its stabilization in liver in response to cAMP (13). This segment also contains specific elements that are responsible for its turnover in LLC-PK₁-F⁺ cells. The latter conclusion was derived by measuring the turnover of a stably transfected chimeric -globin construct that contains the 3'-nontranslated region of the PCK mRNA (11). Such elements could potentially mediate the effect of PMA on PCK mRNA stability in the LLC-PK₁-F⁺ cells.

In contrast to the destabilizing effect of PMA, staurosporine was found to increase the rate of transcription of the PCK gene. Staurosporine was originally thought to be a specific inhibitor of protein kinase C. The observation that the two effectors act through separate mechanisms clearly indicates that the primary action of staurosporine in LLC-PK₁-F⁺ cells is not to inhibit protein kinase C. Staurosporine can also inhibit other serine and tyrosine kinases (29), and it can increase intracellular calcium levels (30). An understanding of how staurosporine causes an increase in transcription of the PCK gene will require further characterization. Through the use of block mutations of specific elements in the PCK₄₉₀CAT construct, the stimulatory effect of staurosporine was mapped to the CRE-1 and P3(II) elements of the PCK promoter. Identical results were obtained when the same set of constructs was used to map the effects of cAMP (18) and of acidic medium (15). Thus all three stimulators of transcription of the PCK gene in LLC-PK₁-F⁺ cells utilize the same set of promoter elements and may utilize the same set of transcription factors.

In hepatoma cells, CREB binds to the CRE-1 element and synergizes with other factors which bind to an upstream "liver specific" region that contains the P3(I), P3(II), and P4 sites (26). The external sites of this region apparently bind a CAATT/enhancer binding protein (C/EBP) (25), whereas the internal P3(II) element binds c-Jun homodimers or c-Jun/c-Fos heterodimers (10, 27). The synergistic interactions of all three transcription factors are required for optimal stimulation of transcription of the PCK gene by cAMP in hepatoma cells. In contrast, in LLC-PK₁-F⁺ cells, C/EBP apparently binds to the CRE-1 site, whereas an unidentified transcription factor occupies the P3(II) site (Ref. 18; and X. Liu and N. P. Curthoys, unpublished data). These findings are consistent with previous footprinting experiments which demonstrated that nuclear extracts from rat liver contain proteins which bind to all four sites (28). However, nuclear extracts from rat kidney produce slightly different footprints of the CRE-1 and P3 sites and do not protect the P4 site.

The addition of staurosporine to confluent cultures of LLC-PK₁-F⁺ cells produces a more pronounced effect on the levels of PCK mRNA than the addition of cAMP or the transfer to acidic medium. In subconfluent cultures, the effects of staurosporine and of cAMP are additive. These results suggest that the effect of staurosporine is mediated by a separate signal transduction pathway. Lines of transgenic mice that express various chimeric PCK- promoter/bovine growth hormone genes have been developed (24). Selective mutation of the CRE-1 element within the transgene causes a 20-fold increase in the renal expression of the growth hormone mRNA compared with the level observed with the wild-type construct. This observation suggests that the endogenous factor that binds to the CRE-1 element in kidney cells may act as both a suppressor and an activator of transcription. Suppression could result from phosphorylation of a unique site on the transcription factor. Staurosporine may act to inhibit the kinase that is responsible for the phosphorylation that inhibits expression of the PCK gene. cAMP, acting through protein kinase A, and decreased pH, acting potentially through a stress-activated MAP kinase, could produce additional phosphorylations that activate the same transcription factor. Thus further efforts to identify the site of staurosporine action in LLC-PK₁-F⁺ cells may help to characterize the mechanism that determines the basal levels of PCK mRNA and to identify the specific factors that mediate both the cAMP- and the pH-responsive induction of the PCK gene within the renal proximal tubule.