THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 267, No. 23, Issue of August 15, pp. 16056-16060. 1992 Printed in LI. S. A.

Expression of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Can Be Regulated by Protein Kinase C*

(Received for publication, January 16, 1992)

Joachim BargonSII , Bruce C. TrapnellS, Kunihiko YoshimuraS, Wilfried DalemansQ, Andrea PaviraniQ, Jean-Pierre LecocqQ, and Ronald G. CrystalS11 From the PPulmonarv Branch. National Heart. Lune. and Blood Institute, National Institutes of Health, Bethesda, Maryland

I

20892 and §Transge& S.A., 67082 Strasbourg, France

Epithelial cells utilize at least two types of apical C1- channels, the CAMP-activated cystic fibrosis trans- membrane conductance regulator (CFTR) and the Ca2+/ calmodulin-dependent C1- channel. While phorbol ester (PMA) activates only CFTR-dependent C1- secretion and the Ca2+ ionophore A23187 only the Ca2+/ca1mod- ulin-dependent C1- secretion, PMA and A23187 share the ability to down-regulate expression of the CFTR gene at the transcriptional level. Since both PMA and A23187 can activate protein kinases, we hypothesized that protein kinase pathways may be involved in the regulation of CFTR gene expression. Exposure of HT- 29 human colon carcinoma cells to the protein kinase C activator SC9 down-regulated CFTR mRNA levels in a dose-dependent fashion, similar to that seen with PMA. The reduction in CFTR transcript levels by SC9 and PMA was blocked by H7, an inhibitor of protein kinases. In a similar fashion, the down-regulation of CFTR transcript levels by A23187 was blocked by H7 as well as staurosporine, another protein kinase inhib- itor. Interestingly, both H7 and staurosporine them- selves increased CFTR mRNA levels. Quantification of CFTR gene transcription rate showed a reduction by SC9 (similar to that with PMA and A23187) that was prevented by H7 and that H7 by itself increased CFTR transcription. Together, these observations suggest that protein kinase pathways, likely including protein kinase C, are involved in the regulation of CFTR gene expression, with activation or inhibition of protein kinase activity down-regulating or up-regulating CFTR gene expression, respectively.

Cystic fibrosis (CF),’ one of the most common fatal hered- itary disorders of Caucasians, is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1-5). These mutations render epithelial cells unable to secrete C1- in response to increased intracellular levels of CAMP (6-9). Recent evidence suggests that the CFTR protein is itself a C1- channel that normally responds to increased intracellular CAMP-activated protein kinase by increasing the

* This work was supported in part by YAssociation Francaise de Lutte contre la Mucoviscidose. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7l To whom reprint requests should be addressed Bldg. 10, Rm. 6D03, National Institutes of Health, Bethesda, MD 20892.

11 Supported in part by the Deutsche Forschungsgemeinschaft. The abbreviations used are: CF, cystic fibrosis; CFTR, cystic

fibrosis transmembrane conductance regulator; PMA, phorbol 12- myristate 13-acetate; kb, kilobase(s); GAPDH, glyceraldehyde-3- phosphate dehydrogenase.

apical membrane C1- permeability (6, 8-15). Epithelial cells also have a distinct apical membrane C1- channel that is activated by Ca2+/calmodulin-dependent protein kinase, but not CAMP-activated protein kinase (16-19). This Ca2+-acti- vated C1- channel is clearly different from the CFTR C1- channel and is functional in epithelia of individuals with CF (16, 17). Although electrophysiologic studies show that the CFTR and the Ca2+-activated C1- channels are independent at the protein level (16-19), there is evidence that the two pathways of epithelial C1- secretion may be interconnected at the level of gene expression (20). In this regard, while increas- ing intracellular Ca2+/Mp2+ levels in epithelial cells increase C1- secretion within minutes, after 3 h there is down-regula- tion in the expression of the CFTR gene at the transcriptional level (20). Likewise, while phorbol myristate acetate (PMA) activates CFTR/cAMP-dependent C1- secretion within min- utes, after 3 h PMA causes epithelial cells to down-regulate CFTR gene expression (21). Since increased intracellular Ca2+ levels as well as PMA activate protein kinases and since protein kinases are involved in the regulation of expression of many genes, we hypothesized that the observed down- regulation of CFTR gene expression by increases in the con- centration of intracellular cation such as Ca2+ and Mg2+ and by PMA may also be mediated by protein kinases. To evaluate this concept, we examined CFTR gene expression in the human colon carcinoma cell line HT-29, an epithelial cell line known to express the CFTR gene, under conditions of acti- vation and inhibition of protein kinases. Interestingly, the data demonstrate that activation of protein kinase(s), prob- ably including protein kinase C, plays a role in CFTR gene regulation.

MATERIALS AND METHODS

Cell Cultures-The human colon carcinoma cell line HT-29 (Amer- ican Type Culture Collection, HTB 38) was maintained in Dulbecco’s modified Eagle’s medium (Whittaker Bioproducts) supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 pg/ml streptomy- cin, and 10% fetal bovine serum with 1.8 mM final ca2+ concentration. For all experiments, cells were evaluated at confluency. Duplicate cultures were incubated for 12 h with media containing from 0 to 100 nM PMA, 0 to 100 p~ N-(6-phenylhexyl)-5-chloro-l-naphthalenesul- fonamide (SC9, an activator of protein kinase C), or the divalent cation ionophore A23187 at the concentration of 2 p M chosen to minimize cellular toxicity (20) (all reagents from Calbiochem). TO inhibit protein kinase activation, 200 pM l-(isoquinolinesu~fonyl)-2- methylpiperazine (H7) or 500 nM staurosporine (both from Calbi- ochem) was added to the culture plates 30 min before the addition of PMA, SC9, or A23187. Under all the conditions used, cell viability was always 295% as measured by trypan blue exclusion.

CFTR mRNA Transcript Leuek-CFTR mRNA transcript levels were evaluated following the various culture conditions by isolating total RNA by the guanidine thiocyanate-CsC1 gradient method (22). Total RNA (10 pg/lane) was evaluated by Northern or slot blot hybridization using a 32P-labeled 4.5-kb human CFTR cDNA probe

16056

Protein Kinase and CFTR Gene Expression 16057

(pTG4976), and mRNA levels were quantitated by densitometric scanning of the autoradiograms as previously described (21). As a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were evaluated in parallel, using a 1.0-kb "'P-labeled human GAPDH cDNA probe (pPB312), encompassing the protein coding sequence (23).

CFTH Gene Transcription Rate-The rate of transcription of the CFTR gene was evaluated by nuclear transcription run-on analysis as previously described (24, 25) , using nuclei isolated from the cells treated under the above described conditions. The DNA targets used were a 4.5-kb human CFTR cDNA (pTG4976), a human c-jun cDNA clone (26), a human genomic c-myc clone (from Lofstrand Labora- tories), and the human [hctin cDNA pHF[fA-1 (27). The plasmid pUC19 containing no human DNA was used as a negative hybridi- zation control and the 4.1-kb 28 S mouse ribosomal RNA gene as a positive control (28). The data were expressed relative to the tran- scription rate of the resting cells (defined as 100%) in each experi- ment.

RESULTS

Modulation of CFTR mRNA Levels-As noted previously (24, 25, 29), HT-29 cells express 6.5-kb CFTR mRNA tran- scripts (Fig. 1, lane 1) . Exposure to 100 nM PMA for 12 h caused a striking reduction in the amount of CFTR transcripts (lane 2). This reduction of CFTR mRNA was completely blocked when cells were preincubated with the protein kinase blocker H7 prior to addition of PMA (lane 3). Although H7 inhibits several protein kinases (30), since PMA specifically activates protein kinase C (31-33) and H7 blocked the PMA- induced down-regulation of CFTR gene expression, it is pos- sible that protein kinase C activation would in itself down- regulate CFTR transcript levels. To evaluate this possibility, SC9, a protein kinase C activator (34,35), was used with and without preincubation of H7. Importantly, an identical pat- tern was observed; SC9 down-regulated CFTR mRNA levels, and this effect was blocked by H7 (lanes 4 and 5 ) . Interest- ingly, H7 alone caused an increase in CFTR mRNA levels (lane 6 ) , suggesting that general base-line inhibition of pro- tein kinase activity up-regulated CFTR gene expression. As a control, under the same conditions, GAPDH mRNA levels in

+ + H7 H7

Rest PMA PMA SC9 SC9 + + + +

CFTR

m

GAPDH

U *

+ ~7

kb

@ +6.5

1 2 3 4 5

+ 1.5

6

FIG. 1. Northern analysis demonstrating PMA- and SC9- induced down-regulation of CFTR gene expression in HT-29 colon carcinoma cells and prevention of this down-regulation by H7. Lane 1, resting HT-29 cells express 6 5 k b C F T R a n d 1.5-kh (;AI'DH mRNA transcripts; lane 2, HT-29 cells after 12-h exposure to 100 nM PMA; lane 3, same as lane 2, but in the presence of 200 pM H7; lane 4, HT-29 cells after 12-h exposure to 100 pM s c 9 ; lane 5 , same as lane 4 hut in the presence of 200 p~ H7; lane 6, HT-29 cells after 12-h exposure to 200 p~ H7.

0 20 40 60 80 100 PMA (nM1

t B.

I\ 0 20 40 60 80 100

SC9 (vM1

Rest + PMA H7 SC9 H7 H7

PMA s c 9

FIG. 2. Dose dependence of the down-regulation of CFTR mRNA transcript levels in HT-29 cells by PMA and the pro- tein kinase C activator SC9 and prevention of this down- regulation by H7, an inhibitor of protein kinases. A, dose- dependent reduction of CFTR mRNA transcript levels by PYA. The dose-response effect of PMA on CFTR mRNA transcript levels was evaluated by slot-blot hybridization using a 12-h incubation and PMA concentrations from 0 to 100 nM. R, dose-dependent reduction of CFTR mRNA transcript levels by SC9. The dose-response effect of SC9 on CFTR mRNA transcript levels was evaluated as above using a 12-h incubation and SC9 concentrations from 0 to 100 phi. C, prevention of the PMA- and SC9-mediated down-regulation of CFTR mRNA levels by HT. Shown are CFTR mRNA levels after 12 h of incubation with 200 p~ H7 alone or in the presence of PMA or s c 9 . For all panels, each data point represents the average of four individ- ual determinations in two separate duplicated experiments.

the HT-29 cells were unchanged by exposure to SC9, PMA, or H7 (lanes 1-6).

Quantitative evaluation of CFTR mRNA transcript levels demonstrated that the effect of PMA was clearly dose-de- pendent (Fig. M) , as was the effect of SC9 (panel B ) . Quan- titative analysis also showed that H7 was capable of blocking both the PMA- and SC9-induced down-regulation of CFTR mRNA transcript levels ( p < 0.01, both comparisons') and that H7 by itself caused a mild up-regulation of CFTR mRNA levels (panel C, p < 0.01).

Interestingly, down-regulation of CFTR mRNA transcript levels by A23187 (Fig. 3A, lanes 1 and 2 ) could also be completely blocked by preincubation with the protein kinase blocker H7 (lane 3). As a control, GAPDH mRNA levels remained unchanged after A23187 or H7 + A23187 exposure. As further evidence that the A23187-induced down-regulation of CFTR gene expression was mediated through protein kinases, another protein kinase inhibitor, staurosporine, was added to the cells prior to the addition of A23187. Importantly, staurosporine blocked the down-regulation of CFTR mRNA levels by A23187 (lane 4 ) . Staurosporine alone resulted in mild up-regulation of CFTR mRNA levels similar to that observed with H7 (lane 5) . Quantitative slot blot assessment (panel 23) of the effects of A23187, H7 + A23187, and H7 alone was similar to that observed by Northern analysis (panel A , rest versus A23187, p < 0.01; A23187 versus H7 +

All data are means k S.E.; all statistical comparisons were made using the two-tailed Student's t test.

16058 Protein Kinase and CFTR Gene Expression

A.

hb

m + 6 5

2 3

1 4

1 A?3187 H i H: siaurO stauro

. . A23187

SOQ11W SPOllne

~23187

FIG. 3. Modulation of A23187-induced down-regulation of CFTR mRNA levels in HT-29 cells by inhibitors of protein kinases. A , Northern analysis demonstrating A2.7187-induced down- regulation of CFTR mRNA levels in HT-29 cells and the prevention of this down-regulation by H7 or staurosporine. Lane I, resting HT- 29 cells; lane 2, HT-29 cells after 12-h exposure to 2 p M A23187; lane :I, same as lane 2, hut in the presence of 200 p~ H7; lane 4 , same as lone 2, but in the presence of 500 nM staurosporine; lane 5 , after 12- h exposure to 500 nM staurosporine. R, quantification of down- regulation of CFTR mRNA levels by A23187 and prevention of this down-regulation by H7 or staurosporine and effect of H7 or stauros- porine alone on CFTR mRNA levels. The experiments were carried out as described for panel A , but quantification was determined by slot-blot hybridization. Each data point represents the average of four individual determinations in two separate duplicated experi- ments.

A23187,p < 0.01; rest versus H7 and rest versus staurosporine, p < 0.01).

Effect of Protein Kinase Activity on CFTR Gene Transcrip- tion Rate-Based on the knowledge that the major effect of PMA and A23187 on the down-regulation of CFTR mRNA expression is at the transcriptional level (20, 24), we hypoth- esized that the effect of these agents on CFTR gene transcrip- tion could be blocked by protein kinase inhibitors and dupli- cated by protein kinase activators. As has been previously described (20,24), PMA and A23187 down-regulated the rate of CFTR gene transcription (Fig. 4; rest versus PMA and rest versus A23187, p < 0.01). SC9, the protein kinase activator, had a similar effect (p < 0.01). Importantly, preincubation of cells with the protein kinase blocker H7 completely prevented the reduction of the transcription rate induced by these agents (A23187, PMA, or SC9 versus H7 + A23187, PMA, or SC9, p < 0.01), while H7 alone up-regulated the CFTR gene tran- scription rate (p c 0.02).

DISCUSSION

The major pathophysiologic consequences of CF emanate from a loss of normal function of the CAMP-dependent/CFTR apical membrane C1- secretory pathway of epithelial cells (1,

H S I * 1 I * + + I

PMA H7 SC9 H7 A23187 H7 H7

PMA SC9 A23187

FIG. 4. Down-regulation of the CFTR gene transcription rate by PMA, SC9, and A23187 and prevention of this down- regulation by H7. The CFTR gene transcription rate was evaluated by nuclear run-on analysis and compared with that of the genes for c-jun, c-m.vc, 6-actin, pUC19 (an irrelevant plasmid), and ribosomal RNA. Shown is a comparison of the relative transcription rate of the CFTR gene from resting HT-29 cells to the transcription rate of cells in the presence of 100 nM PMA, 100 PM SC9, and 2 pht A2.7187- treated cells in the presence or absence of H i and effect of H i alone. The data represent the average of three separate experiments.

6-10, 36). Epithelial cells also have an independent apical pathway of C1- secretion that is activated by Ca’+-dependent calmodulin (16, 19). Previous studies have shown that PMA, an agent that activates the cAMP/CFTR pathway to secrete C1- within minutes, over several hours down-regulates the expression of the CFTR gene by suppressing the rate of CFTR gene transcription (21, 24). Interestingly, agents that activate the Ca’+/calmodulin pathway to secrete C1- in minutes also down-regulate CFTR gene transcription over hours, suggest- ing that despite the independence of the two C1- channels at the protein level, control of the two pathways is linked at the level of CFTR gene regulation (16-20).

The present study brings these disparate observations to- gether by demonstrating that the down-regulation of CFTR transcription by PMA and increased intracellular Ca” levels are both mediated by activation of protein kinase(s), including protein kinase C. Further, inhibition of protein kinases causes an up-regulation of CFTR gene expression, suggesting protein kinase pathways can have significant influence on the extent of CFTR gene expression.

Protein Kinases and CFTR-With the identification of the CFTR gene came the prediction that the putative protein product had recognition sites that could be phosphorylated, and it was hypothesized that protein kinases might modulate the function of the CFTR protein (2). This was consistent with the prior observation that activators of protein kinases increase C1- secretion in epithelial cells of normal individuals but not those of individuals with CF (7-10). Likewise, the addition of protein kinase A or C to apical membranes of normal epithelial cells activates C1- secretion but has no effect on apical membrane C1- secretion of CF cells (7-10). Recent studies suggest that protein kinases have a role in this process by phosphorylation of the R domain of the CFTR protein (37, 38).

The observations in the present study demonstrate that protein kinases can also play an important role in the regu- lation of CFTR gene expression by modulating the rate of CFTR gene transcription. We made this observation by in- vestigating the pathways by which phorbol esters and modu- lators of intracellular Ca2+ levels down-regulate CFTR gene transcription. It is not surprising that PMA modulates CFTR gene expression as the CFTR 5”flanking region contains at least two types of PMA-responsive elements, the binding sites for the transcription factors AP-1 and AP-2 (24,39,40). PMA serves as an analogue of diacylglycerol in its capacity to

Protein Kinase and CFTR Gene Expression 16059

activate protein kinase C, and protein kinase C activation has been implicated in the regulation of several genes (32,33,41). The concept that protein kinase C is likely involved is sup- ported by the ability of protein kinase blockers such as H7 or staurosporine to block the down-regulation of CFTR gene expression by PMA. For example, H7 inhibits protein kinase C by interacting with the catalytic site of the enzyme, although H7 also can inhibit other protein kinases through similar interactions (30). The same is true for staurosporine, an agent that blocks a variety of protein kinases, including protein kinase C, CAMP-dependent protein kinase and Ca*+/calmod- ulin-dependent protein kinase (42,43). However, the specific- ity for protein kinase C in the event of down-regulation of CFTR gene expression is demonstrated by the use of specific activators. For example, PMA is quite specific for activating protein kinase C with no effect on protein kinase A or Ca2+/ calmodulin-dependent protein kinase (31). Similarly, SC9, another potent activator for protein kinase C as a substitute for phosphatidylserine without significant effect on either Ca’+/calmodulin-dependent protein kinase, CAMP-dependent protein kinase 11, or cGTP-dependent protein kinase (34,35) can also suppress CFTR gene expression, and the effect of SC9 on CFTR gene expression is blocked by H7. Further, intracellular Ca2+ mobilization is known to activate Ca2+/ calmodulin-dependent protein kinases (18,44) and to regulate the expression of some genes, likely through other signal transduction mechanisms (45). This is also true for the CFTR gene, since ionophores that mobilize intracellular divalent cations such as A23187 or ionomycin down-regulated the expression of the CFTR gene at the transcriptional level (20). The observations that H7 as well as staurosporine block A23187-induced down-regulation of CFTR gene expression implicate an active role of protein kinases in this pathway. Although A23187 could also activate the regulatory pathway for CFTR gene expression by a change in intracellular pH, it appears that protein kinase(s) is still the intermediate due to blockage by appropriate protein kinase inhibitors as discussed above. Finally, H7 by itself increased the rate of CFTR gene transcription. Together, this evidence strongly implicates that protein kinases, including protein kinase C, are involved in intracellular signaling pathways that modulate the rate of CFTR gene transcription. Thus, protein kinases play two general roles in the regulation of CFTR the transcription rate of the CFTR gene and the activation of the CFTR protein to secrete C1-.

Clinical Implications of Protein Kinase Regulation on CFTR Gene Expression-The observation of the links of protein kinase pathways and CFTR gene expression has several im- plications with potential importance for the development of new therapeutic strategies for CF. First, it has been suggested that it may be possible to circumvent the defective CAMP/ CFTR-dependent C1- secretory pathway if a suitable agent could be found to stimulate the Ca2+-dependent C1- secretory pathway (6,18). The observations in the present study suggest that if such a therapeutic agent worked through protein kinases that were relevant to CFTR gene transcription, an added “bonus” of this strategy would be to down-regulate the expression of the abnormal CFTR gene. This concept is consistent with the suggestion that the absence of CFTR may be associated with less severe pulmonary disease than the common AF508 mutation (46-48). Second, in the context that the protein product of the common AF508 CFTR mutation will function to secrete C1- (49, 50) and in the context that there are other CFTR mutations with partial CFTR function (51), it may be useful to up-regulate the amount of the partially functional CFTR protein. In this regard, an inter-

esting observation of this study is the up-regulation of CFTR transcription rate and CFTR mRNA levels by protein kinase blockers.

These observations may also have implications for the understanding of the pathophysiology of individuals with normal CFTR alleles but with airway inflammation. Because PMA is a model “inflammatory” stimulus (32, 52) and acti- vation of protein kinase C as well as increases of intracellular Ca2+ levels are common second messengers in activated cells, it is conceivable that various inflammatory stimuli might also down-regulate CFTR gene expression in normal epithelial cells, such that they cannot secrete C1- in a normal fashion.

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