©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for Involvement of Phosphatidylcholine-Phospholipase C and Protein Kinase C in Transforming Growth Factor- Signaling (*)

Jennifer Halstead , Kathleen Kemp , Ronald A. Ignotz (§)

From the (1) Department of Cell Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor- (TGF-) is a multifunctional peptide that elicits a wide variety of responses in cells. TGF- binds to cell surface receptors that contain cytoplasmic serine/threonine kinase domains. Here we provide evidence that both phospholipase C and protein kinase C (PKC) are involved in the TGF- activation of transcription and luciferase expression from the p3TP-Lux plasmid. Down-regulation of PKC prevents TGF-1 induction of luciferase expression. Staurosporin and Calphostin C, inhibitors of PKC, block the ability of TGF-1 to initiate transcription of the luciferase gene. Further, D609, an inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), and secondarily PKC also blocks TGF-1-induced transcription of the transgene in A549 cells while the phosphatidylinositol-PLC pathway inhibitor U73122 is without effect. TGF- elevates steady-state mRNA levels for the endogenous PAI-1 and fibronectin genes. Treatment of cells with calphostin C or D609 prevents the TGF--induced increase in these mRNAs. Together, these results suggest that PC-PLC and PKC are in a TGF- signaling pathway that results in elevated gene expression.


INTRODUCTION

TGF-() has been implicated in activities ranging from embryogenesis and differentiation to promoting wound healing in vivo. In vitro, TGF- enhances extracellular matrix production as well as cell adhesion, inhibits the proliferation of epithelial and hematopoietic progenitor cells, acts as an immunosuppressive agent, and modulates the differentiation of a variety of cell types including adipocytes, keratinocytes, myoblasts, and hematopoietic progenitors (1, 2) . Growth suppression has been correlated to down-regulation of c-myc expression (3) , blockade of the phosphorylation of pRb (retinoblastoma gene product) in late G(4) , and inhibition of cyclin-dependent kinases (5, 6, 7) . However, the steps between receptor binding and these nuclear events remain obscure. TGF- initiates responses through binding to cell surface receptor proteins (type I and type II receptors) that contain serine/threonine kinase motifs in their cytoplasmic domains (8) . This implies initiation of a phosphorylation cascade as early steps in receptor signaling. Several lines of evidence indicate that type I and type II receptors form heterodimeric signaling complexes (9, 10, 11) . Recently, the Massagué laboratory (12) reported that TGF- binds to the type II receptor, which then recruits and phosphorylates the type I receptor on its cytoplasmic domain. The type II receptor appears to be constitutively phosphorylated. In addition, homodimers of type II receptors have been observed in the absence and presence of TGF- (13) . The nature and identity of additional intermediates in the early postreceptor signal transduction pathways utilized by TGF- remain to be elucidated. Here we provide evidence that both phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) may be early intermediates in TGF- signaling pathways that lead to activation of gene transcription.


MATERIALS AND METHODS

The human lung carcinoma cell line, A549, was obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and at 37 °C and 5% CO. Porcine TGF-1 was obtained from R& Industries, Minneapolis, MN. Pathway inhibitors and toxins were obtained from Calbiochem with the exception of U73122, which was from Biomol Inc., Plymouth Meeting, PA. Luciferase assay kit was purchased from Promega, Madison, WI. Luciferase activity was measured using an Analytical Luminescence Laboratory Monolight 2010 luminometer. Emitted light was measured over a 30-s interval. Chloramphenicol acetyltransferase (CAT) activity was assessed by thin layer chromatography and scanning of plates on a Betagen Betascope 603 blot analyzer. For transfection, A549 cells were plated at 1.5 10 cells/100-mm plate and allowed to grow overnight. The next morning cells were co-transfected with 15 µg/plate of the p3TP-Lux plasmid and 2 µg of pCAT-control plasmid (Promega) via the DEAE-dextran method (14) . Briefly, cells were washed 3 times with phosphate-buffered saline followed by incubation with plasmid DNA, 100 µg/ml DEAE-dextran, and 4 µM chloroquine in serum-free DMEM. After a 3-h incubation, cultures were treated with 10% glycerol in serum-free DMEM for 90 s followed by washing with phosphate-buffered saline. Transfected cells were allowed to recover for 24 h prior to induction. All subsequent treatments were done in serum-free DMEM supplemented with 100 pM TGF-1 and various inhibitors. Cells were extracted in reporter lysis buffer (Promega) to permit analysis of both luciferase activity and CAT activity.

For Northern blotting and analysis of steady-state mRNA levels for PAI-1 and fibronectin (FN), confluent cultures of A549 cells were treated with the various test agents for either 3 h (PAI-1) or 5 h (FN). Total cellular RNA was extracted using the Trizol reagent (Life Technologies, Inc.) following the manufacturer's protocols. Fifteen micrograms of total cellular RNA were fractionated on 1% agarose gels containing formaldehyde followed by transfer to Boehringer Mannheim nylon membranes. The membranes were hybridized to P-labeled probes for human PAI-1 or rat fibronectin. The PAI-1 probe (PAI-1 oligonucleotide probe from Oncogene Sciences) was end-labeled with P using T4 polynucleotide kinase. The FN cDNA probe was labeled by the random prime method using a kit from DuPont NEN.


RESULTS AND DISCUSSION

A549 human lung carcinoma cells are growth-inhibited by TGF- (15). We have used this cell line to begin to address TGF- signal transduction. A TGF--responsive promoter linked to the luciferase gene, the p3TP-Lux plasmid, obtained from Dr. Joan Massagué (10) was utilized as a marker for activation of gene expression. The p3TP-Lux plasmid consists of the TGF- response element from the PAI-1 gene and three tandemly arranged phorbol ester response elements (TREs) placed 5` to the luciferase gene. This plasmid is responsive to both TGF- and phorbol esters.

Following transfection, A549 cells were placed into serum-free DMEM and treated with 100 pM TGF-1. A kinetic analysis (Fig. 1A) of the response of the transgene to TGF-1 indicates a rapid initiation in transcription with an approximately 2-fold elevation in luciferase activity within 1 h. Luciferase activity continued to rise to 8-10-fold at 6 h and approximately 20-40-fold at 18-20 h (not shown).


Figure 1: A, kinetic analysis of TGF- response. Transfected A549 cells were transferred to serum-free DMEM supplemented with 150 pM TGF-1. At the indicated times cells were harvested and processed for luciferase analysis and CAT activity using the Promega kit according to the manufacturer's protocols. The results shown are the average of triplicate cultures and normalized for CAT activity and protein content. TGF- induces a rapid (within 2 h) elevation in luciferase expression that continues to rise to approximately 10-fold by 6 h and over 20-fold by 18 h (not shown). B, down-regulation of PKC blocks TGF- activation. Transfected A549 cells were treated with 200 ng/ml PMA (in DMEM with 10% fetal bovine serum) for 40 h to down-regulate PKC. The medium was replaced with serum-free DMEM supplemented with 100 pM TGF-1 or 100 ng/ml PMA. After a 3-h incubation the cells were harvested, extracts were prepared, and luciferase activity was determined. The results shown represent the average of triplicate samples and are normalized for protein content. Both TGF-1 and PMA induce a 3-5-fold induction of luciferase activity in parallel control cultures, but following PKC down-regulation, neither TGF-1 nor PMA could induce expression of the transgene.



To address the role of PKC in TGF- signaling, transfected A549 cells were treated with 200 ng/ml phorbol myristate acetate (PMA) for 40 h to down-regulate PKC (16) . For induction, medium was replaced with serum-free DMEM alone or supplemented with 100 pM TGF-1 or 100 ng/ml PMA. Following a 3-h incubation, cell extracts were prepared and assayed for luciferase activity. Fig. 1B shows that in control cultures in which PKC was not down-regulated both TGF-1 and PMA induce elevated luciferase activity. In contrast, following down-regulation of PKC, neither TGF-1 nor PMA was capable of inducing expression of the transgene.

Simultaneous treatment of transfected cells with increasing concentrations of the PKC inhibitor, staurosporin (Fig. 2A), and 100 pM TGF-1 result in a nearly complete inhibition of luciferase activity by the highest concentration of staurosporin tested. Half-maximal inhibition was observed at approximately 50 nM staurosporin. A second inhibitor of PKC, calphostin C, also inhibited the TGF- response, although a higher concentration was required to obtain maximal inhibition. Calphostin C binds to the regulatory subunit of PKC while staurosporin appears to interact with the ATP-binding site (17, 18). This difference in mode of action or differences in drug stability and half-lives may account for varied effectiveness of the drugs. The ability of PKC inhibitors to block TGF-1-induced gene expression together with the lack of response following down-regulation of PKC suggests that protein kinase C is a likely early downstream mediator of TGF- responses, which result in altered gene expression.


Figure 2: Antagonists of protein kinase C inhibit TGF- response. p3TP-Lux-transfected cells were treated simultaneously with 100 pM TGF-1 and the indicated concentrations of either staurosporin (A) or calphostin C (B). After 3 h, cells were harvested and processed as described in the legend to Fig. 1. The results shown are the average of determinations made from triplicate cultures. The dashedlines indicate the level of luciferase activity in uninduced cultures.



Diacylglycerol (DAG), the endogenous activator of PKC, is generated by the hydrolysis of phospholipids. We examined the ability of a newly described inhibitor of phospholipase C, D609, to block TGF-'s ability to activate transcription of the luciferase gene. D609 blocks PC-PLC (19) and secondarily inhibits the activation of PKC by preventing the generation of DAG. p3TP-Lux-transfected A549 cells were preincubated in serum-free medium with the indicated concentrations of D609 for 1 h. 100 pM TGF-1 was added, and the incubation was continued for an additional 4 h after which time cells were harvested, processed, and assayed for luciferase activity. The results shown in Fig. 3A indicate that D609 treatment resulted in half-maximal inhibition at 6-7 µg/ml and complete inhibition of TGF--activated transcription of the transgene at 25-50 µg/ml. U73122 (20) , an inhibitor of phosphatidylinositol-PLC-mediated responses, was incapable of blocking TGF--activated luciferase expression (Fig. 3B). We have consistently observed that U73122 treatment can augment the TGF- induction of luciferase. Thus, direct inhibition of protein kinase C via either down-regulation or staurosporin or calphostin C treatment as well as indirect inhibition of protein kinase C via D609 block TGF-1-induced transcription from the p3TP-Lux plasmid. These results imply that both PC-PLC and PKC are important intermediates in the generation of a signal by TGF-.


Figure 3: D609, a phosphatidylcholine-specific phospholipase C inhibitor blocks TGF- responsiveness. A, transfected A549 cells were pretreated for 1 h in serum-free DMEM with the indicated concentrations of D609. 100 pM TGF-1 was added and the incubation continued for an additional 4 h. The cells were harvested, extracted, and assayed for protein content and luciferase activity as described under ``Materials and Methods.'' The results shown are the average of triplicate cultures and indicate that D609 completely inhibits the TGF- effect. The dashedline indicates the level of luciferase activity in uninduced cultures. B, transfected A549 cells were treated with the indicated concentrations of U73122 and 100 pM TGF-1. Following a 4-h incubation, the cultures were harvested and assayed for luciferase and CAT activity. The results are the averages of duplicate cultures.



The above results are in contrast to the results obtained using inhibitors of G proteins in this assay. Neither cholera toxin nor pertussin toxin was capable of inhibiting TGF-1 activation of transcription of the p3TP-Lux plasmid (not shown). Cholera toxin inhibits G while pertussis toxin inhibits G, both regulators of adenylate cyclase activity. We conclude that PC-PLC and protein kinase C are important intermediates in TGF- signaling pathways, but toxin-inhibitable G proteins (G and G) are not essential for TGF- activation of transgene expression in A549 cells. Others have reported that these G proteins may be involved in TGF- stimulation of proliferation of AKR-2B fibroblasts (21, 22, 23) suggesting that different cell types and responses may utilize different signaling pathways.

The observation that specific antagonists can block the expression of a transgene in response to TGF- can be extended to endogenous genes encoding PAI-1 and fibronectin. Expression of both genes as measured by steady-state mRNA levels is elevated by TGF-1 treatment. Co-treatment with TGF-1 and either calphostin C or D609 blocks the elevation in PAI-1 and fibronectin mRNA levels (Fig. 4). Thus, one pathway utilized by TGF- to enhance expression of extracellular matrix genes includes PC-PLC and PKC.


Figure 4: Endogenous PAI-1 and fibronectin gene expression also inhibited. Examination of steady-state mRNA levels by Northern blotting reveals that both calphostin C and D609 inhibit the ability of TGF-1 to elevate mRNA levels for PAI-1 and fibronectin. A, A549 cells were incubated for 3 h in serum-free DMEM containing test agents. Total cellular RNA was isolated and fractionated on 1% agarose gels containing formaldehyde and processed from Northern blotting and detection of PAI-1 mRNA. Lanes: 1, untreated; 2, 100 pM TGF-1; 3, 100 pM TGF-1 + 200 nM calphostin C; 4, 100 pM TGF-1 + 20 µg/ml D609. Following development of the autoradiograph, the blot was stripped in boiling dHO and reprobed for glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA to assess uniformity of RNA between samples. B, A549 cells were treated as above but for 5 h. Following electrophoresis, the agarose gel was incubated in 50 mM NaOH prior to transfer to facilitate efficient transfer of the 7-8-kilobase FN mRNA. The NaOH treatment likely accounts for the diffuse appearance of the signals for FN mRNA and glyceraldehyde-phosphate dehydrogenase mRNA.



Receptor tyrosine kinases also interact with PLC isoforms. These receptors activate phosphatidylinositol-PLC such as PLC- (24) . Hydrolysis of phosphatidylinositol generates DAG, which activates PKC but also produces IP (inositol 3-phosphate), which stimulates release of Ca by binding to IP receptors. Receptor tyrosine kinases through PLC- are capable of activating PKC and affecting Ca-sensitive enzymes and pathways. PC-PLC hydrolyzes PC to yield DAG and phosphocholine (25) ; thus, while PKC is activated, the absence of IP would not result in release of Ca. PC-PLC has recently been shown to be important in signaling by tumor necrosis factor receptors and activation of the transcription factor NF-B (19, 26) . PC-PLC may also be important in the mitogenic response to platelet-derived growth factor and in ras-transformed cells (27, 28) . Diaz-Meco et al.(29) have suggested that TGF- may prevent the coupling of ras p21 to PC hydrolysis, thus contributing to the antiproliferative response of TGF-1.

TGF- utilizes multiple signaling pathways to generate the diversity of responses observed. For example, activation of transcription may involve a different pathway than growth inhibition. Others have reported that TGF- rapidly activates the ras protooncogene (30) and MAP kinase (31) . ras activity may be required for PC-PLC activation (32) , and recently it was reported that MAP kinase may be in a pathway downstream of PKC (33) . Selective activation of pathways by TGF- may be cell type-dependent. Last, overlap exists between signaling pathways utilized by TGF- receptors and receptor tyrosine kinases.


FOOTNOTES

*
This work was supported in part by a grant from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester MA 01655. Tel.: 508-856-6106; Fax: 508-856-5612.

The abbreviations used are: TGF-, transforming growth factor-; DMEM, Dulbecco's modified Eagle's medium; PC-PLC, phosphatidylcholine-specific phospholipase C; PKC, protein kinase C; CAT, chloramphenicol acetyltransferase; PAI-1, plasminogen activator inhibitor-1; FN, fibronectin; PMA, phorbol myristate acetate; DAG, diacylglycerol; IP, inositol 3-phosphate.


ACKNOWLEDGEMENTS

We thank Dr. Joan Massagué for generously providing the p3TP-Lux plasmid. We thank Cindy Richard for assistance in preparation of this manuscript and Dr. Gary Stein for critical reading and discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.