Vol. 12, No. 1

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1992, p. 261-265

0270-7306/92/010261-05$02.00/0 Copyright C 1992, American Society for Microbiology

Evidence For The Involvement of Protein Kinase Activity In Transforming Growth Factor-1 Signal Transduction MASAHIKO OHTSUKI AND JOAN MASSAGUIt* Cell Biology and Genetics Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Received 28 August 1991/Accepted 16 October 1991

Transforming growth factor-Il (TGF-I1) rapidly increases the expression of junB transcription factor and plasminogen activator inhibitor-i (PAI-1) and prevents the cell cycle-dependent phosphorylation of the RB retinoblastoma susceptibility gene product during late G, phase in MvlLu lung epithelial cells. These responses are shown in this report to be blocked by the potent serine/threonine protein kinase inhibitor, H7, added with TGF-131. Added alone, H7 does not alter the basal junB or PAI-1 mRNA levels, the deposition of PAI-1 into the extracellular matrix, or the phosphorylation of RB in late G1 phase, suggesting that this inhibitor does not have a general nonspecific effect on the cell. The analogs H8 and H9, which are preferential inhibitors of cyclic nucleotide-dependent protein kinases, are fivefold less potent than H7 as inhibitors of the TGF-, response. The PAI-1 response to TGF-,1 is not affected by the simultaneous addition of staurosporine, which is a protein kinase C inhibitor, or by the prolonged preincubation of cells with phorbol 12-myristate 13-acetate, which down-regulates protein kinase C. The results suggest the possibility that H7 and its analogs block various early TGF-j responses by inhibiting a protein serine/threonine kinase(s). Regulation of gene expression by TGF-p could involve RB. This possibility is suggested by the observation that RB interacts with the E2F transcription factor (2, 3, 6, 7), particularly when RB is in the underphosphorylated form (3, 6). Indeed, RB appears to be involved in down-regulating c-myc transcription by TGF-p1 in keratinocytes (26). This involvement, however, seems less likely in other cells, including MvlLu cells (12, 16) and the DU145 human carcinoma cell line (35), which is deficient in RB function (4). TGF-p may also regulate gene expression through CREB, a transcription factor whose phosphorylation is rapidly increased by TGF-pl (14). The phenotype of certain TGF-p-resistant MvlLu cell mutants suggests that the initiation of multiple TGF-p responses in these cells depends on its interaction with two high-affinity membrane components known as TGF-P receptors I and II (5, 17, 18). The primary structure of these proteins and the nature of their signalling mechanism have not been described yet. However, the deduced structure of a recently cloned activin receptor suggests that this protein could be a ligand-activated serine/threonine protein kinase (22). This raises the important possibility that one or several protein kinases may play central roles in signal transduction by activin and other factors of the TGF-p family. Here, we report results of studies designed to investigate the possibility that a protein kinase activity may be involved in mediating TGF-p effects. The results show that several early gene responses and growth-inhibitory effects of TGF-1 in MvlLu cells are mediated by events that can be potently blocked by H7 and related protein kinase inhibitors.

Transforming growth factor-p (TGF-P) belongs to a family of growth and differentiation factors that also includes the activins and inhibins, the bone morphogenetic proteins, and the Mullerian inhibiting substance in mammals, as well as other members identified thus far only in amphibians or insects (21, 27). TGF-P, which exists in various isoforms (p1, P2, and P3) that are encoded by closely related genes, can inhibit or stimulate cell proliferation, promote extracellular matrix formation, affect cell differentiation, and regulate other cellular functions. These responses are observed in cells from virtually every lineage, and their intensity may vary with each TGF-p isoform. Early cellular responses to TGF-P include effects on the expression of several genes. The MvlLu lung epithelial cell line, whose proliferative cycle is arrested by TGF-p in late G1 phase (11, 18, 19, 33), responds to TGF-p with elevated expression of the junB transcription factor and the secretory inhibitor of extracellular matrix degradation, plasminogen activator inhibitor-1 (PAI-1) (16, 24). MvlLu cells also respond to TGF-P with decreased c-myc gene expression (35), which could contribute to the growth-inhibitory effect of TGF-P, as has been shown for other cell types (25). The junB, PAI-1, and c-myc responses occur rapidly following addition of TGF-P to MvlLu cells at any stage during the G, phase or S phase of the cell cycle (35). In addition to these effects, TGF-p can prevent phosphorylation of the RB retinoblastoma susceptibility gene product during the late G1 phase (15). This effect is not secondary to cell cycle arrest. TGF-P seems to prevent the activation of a G1-phase-specific RB kinase (15) which might belong to the cdc2 protein kinase family (11). Since cell cycle-dependent phosphorylation may interrupt the ability of RB to function as a negative checkpoint at the end of G1 (20), it is possible that TGF-P causes arrest of the MvlLu cell cycle in late G1 by retaining RB in its underphosphorylated, growth-suppressive state. *

MATERIALS AND METHODS

Reagents. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8), and N-2-(methylamino)-5-isoquinolinesulfonamide dihydrochloride (H9) were obtained from Seikagaku America (Rockville, Md.). Staurosporine was from Kamiya Biochemical Company

Corresponding author. 261

262

OHTSUKI AND MASSAGUE

(Thousand Oaks, Calif.). Bovine TGF-31 and TGF-P2 were purified as previously described (30). Phorbol 12-myristate 13-acetate (PMA) was from Sigma Chemical Co. Cell culture. The MvlLu mink lung epithelial cell line (CCL-64; American Type Culture Collection) was maintained in minimal essential medium (GIBCO) containing nonessential amino acids and 10% fetal bovine serum. Northern (RNA) blot assays. Total RNA was isolated with RNAzol B (Biotex Laboratories, Houston, Tex.), separated on 1% agarose-morpholinepropanesulfonic acid (MOPS)formaldehyde gels, and transferred with a Posiblot (Stratagene, La Jolla, Calif.) to Nytran membranes (Schleicher & Schuell). Specific mRNAs were detected by using as probes cDNAs corresponding to human junB (p465.20 [29], obtained from D. Nathans), human PAI-1 (PAI-lA1 [1], obtained from P. Andreasen), and rat glyceraldehyde-3-phosphate dehydrogenase (pRGAPDH-13 [8]). All probes were labeled with 32P by random priming (Multiprime; Amersham) to specific activities over 109 dpm/,ug. Hybridization signals were detected by autoradiography or recorded on a phosphor screen and scanned with a Phosphorimager 400-E (Molecular Dynamics, Sunnyvale, Calif.) set to detect 32P radioactive emissions over a 5-order linear range of sensitivity. Metabolic labeling of extracellular matrix proteins. Cells in methionine-free minimal essential medium received the indicated concentrations of inhibitors 5 min prior to addition of TGF-,1 (250 pM) or PMA (10 ng/ml) and were incubated for 4 h. Tran35S-label (40 ,uCi; ICN Biochemicals) was added during the final 2 h of this incubation. Labeled cell extracts enriched for extracellular matrix proteins were prepared by selective extraction with detergents as previously described (9, 16) and analyzed on 8 or 10% polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate. Gels were fixed and fluorographed with Enlightening (New England Nuclear). Signals were recorded and scanned with a Phosphorimager 400-E. Radioactive PAI-1 signals were digitized and quantified with the Image Quant Software (Molecular Dynamics). RB protein assays. MvlLu proteins were extracted, quantitated, and subjected to Western immunoblot analysis with anti-RB monoclonal antibody (G3-245; PharMingen) and alkaline phosphatase-conjugated second antibody (Sigma) as previously described (15). RESULTS Inhibition of early gene responses to TGF-II1 by protein kinase inhibitors. The early responses of MvlLu cells to TGF-P include an increase in junB and PAI-1 mRNA levels (16, 24, 35). To investigate whether a protein kinase might be involved in the mediation of any of these responses, MvlLu cells were stimulated with TGF-pl in the presence of several protein kinase inhibitors. H7 is an inhibitor of protein kinase C (Kg, 6.0 ,uM), protein kinase A (Ki, 3.0 ,uM), protein kinase G (Ki, 5.8 ,uM), and other protein kinases (10). The related compounds H8 and H9 are more potent as inhibitors against protein kinase A (Ki, 1.2 and 1.9 ,uM, respectively) and protein kinase G (Ki, 0.5 and 0.9 ,uM, respectively) than against protein kinase C (Ki, 15 and 18 ,uM, respectively) (10). H7, H8, or H9 was added to exponentially growing cells 5 min before stimulation of these cells with TGF-,1l. For comparison, parallel incubations were carried out with PMA, which also induces a rapid elevation of junB and PAI-1 mRNA levels in these cells and acts through protein kinase C (23). The elevation ofjunB and PAI-1 mRNA levels

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induced by a 90-min exposure of cells to TGF-pl or PMA was completely blocked by 50 ,uM H7 and was strongly inhibited by 100 ,uM H8 or H9 (Fig. 1). Added to MvlLu cells for up to 90 min, H7, H8, or H9 alone did not affect the basal level of junB (as seen by overexposure of the blot shown in Fig. 1 [data not shown]) or PAI-1 mRNA (Fig. 1). The effect of H7 was not due to interference with TGF-p binding to receptors I and II or betaglycan (data not shown), as determined by TGF-1 receptor affinity-labeling assays (5, 15). Effect on PAI-1 protein levels. The effect of protein kinase inhibitors on the TGF-pl response was investigated in more detail by using the rate of extracellular matrix PAI-1 synthesis as a convenient parameter to monitor the cellular response to TGF-,B1 and PMA. PAI-1 was identified as a 44- to 46-kDa doublet by polyacrylamide gel electrophoresis of extracellular matrix extracts (13). The 44-kDa form of PAI-1 is a proteolytic product of the 46-kDa form (13), as confirmed by comparative peptide mapping and immunoprecipitation with anti-PAI-1 antibody (data not shown). TGF-pi and PMA increased the level of both PAI-1 forms. In addition, TGF-f1 and, to a lesser extent, PMA increased the proportion of the larger PAI-1 form (Fig. 2). Treatment of confluent MvlLu cells with H7 prevented these effects of TGF-pi and PMA in a dose-dependent manner (Fig. 2A). The protein kinase inhibitors H8 and H9 also inhibited this response (Fig. 2B), but their potency against TGF-pl (50% effective dose [ED50] = 100 ,iM) and PMA (ED50 > 100 ,uM) was lower than that of H7 (ED50 = 20 ,uM against TGF-pl and ED50 = 65 ,M against PMA), as determined by testing various concentrations of these inhibitors and quantitating the radioactive PAI-1 signal as described in Materials and Methods (data not shown). Similar results were obtained for the effect of TGF-pl or PMA on the level of PAI-1 released into the medium (data not shown). The observed order of potencies of these protein kinase inhibitors as blockers of the PMA effect was consistent with the expected involvement of protein kinase C in the cellular response to PMA. It was also compatible with the involvement of this kinase activity, rather than that of protein kinase A or G, in the response of cells to TGF-31. This possibility

TGF-P SIGNAL TRANSDUCTION

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FIG. 2. Effects of protein kinase inhibitors and protein kinase C depletion on the PAI-1 biosynthesis response to TGF-11 and PMA. Confluent MvlLu cells received H7 at the indicated concentrations (A), 100 ,M H8 or 100 ,M H9 (B), or staurosporin (SP) at the indicated concentrations (C). After 5 min, TGF-p1 (250 pM) or PMA (10 ng/ml) was added as indicated, and incubations continued for 4 h. Cells were metabolically labeled with 15[S]methionine during the final 2 h of incubation. Labeled cell extracts enriched for extracellular matrix proteins were analyzed by gel electrophoresis and fluorography. The positions of the two forms of PAI-1 and the markers of molecular weight (in thousands) are indicated. Cells were incubated with PMA (100 ng/ml) for 16 h (D). They were then incubated for 4 h with 250 pM TGF-11, 250 pM TGF-32, 10 ng of PMA per ml, or no addition. Labeled extracellular matrix extracts were obtained and analyzed as described above.

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TGF-pl under conditions that normally prevent protein kinase C function. In one set of experiments, the response to TGF-,13 and PMA was tested in cells treated with the potent inhibitor of protein kinase C, staurosporine (32). Whereas staurosporine inhibited the PAI-1 response to PMA, it was ineffective against TGF-p1 (Fig. 2C). Moreover, the PAI-1 response to TGF-pl or TGF-P2 was retained in cells that had been desensitized to PMA by prolonged (16-h) preincubation with a high concentration of this agent (Fig. 2D). Prolonged incubation with PMA is known to strongly down-regulate protein kinase C (28). These results argued against the involvement of a PMA-sensitive protein kinase C in TGF-P action. Additional evidence supporting this interpretation was provided by the observation that the maximal effects of TGF-pl and PMA on PAI-1 production were additive (data not shown). Taken together, these results suggested that a kinase activity distinct from that of protein kinase C, A, or G mediated various early gene responses to TGF-pl. Protection against TGF-,1 inhibition of RB phosphorylation. The response of MvlLu cells to TGF-pl is also characterized by an inhibition of RB phosphorylation in late G1 phase of the cell cycle (15). To determine whether this response was affected by protein kinase inhibitors, contact-

inhibited cell monolayers were stimulated to enter the proliferative cycle by being replated at low density in the presence of fresh medium containing 10% fetal bovine serum. After 6 h, the cell population had initiated RB phosphorylation as detected by the appearance of RB forms whose migration on electrophoresis gels was characteristically slow (Fig. 3), as previously described (15, 20). Conversion of RB to phosphorylated forms progressed until it was nearly complete by 12 h. When TGF-p1 was added at the 6-h time point, it prevented the further conversion of RB to highly phosphorylated forms (Fig. 3). When added alone at the 6-h time point, H7, H8, or H9 did not perturb the process of RB phosphorylation. However, these agents completely prevented the inhibition of RB phosphorylation by TGF-pl (Fig. 3). Thus, at least one manifestation of the growth-inhibitory response to TGF-,1l was eliminated by the presence of H7 or related protein kinase inhibitors. Additional experiments were conducted to determine whether the negative effects of TGF-,1l on histone H4 mRNA elevation or on DNA synthesis at the onset of S phase were also susceptible to inhibition by H7. However, such experiments were uninformative, because H7 acting alone had a strong inhibitory effect on these two parameters (data not shown).

OHTSUKI AND

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DISCUSSION The present evidence that a protein kinase is centrally involved in mediating multiple TGF-,B responses in MvlLu cells rests on the observation that H7 and its analogs H8 and H9 completely inhibit these responses. These compounds are isoquinolinesulfonamide derivatives, developed as potent inhibitors of serine/threonine protein kinases, and are devoid of the anticalmodulin activity of naphthalenesulfonamides (10). The activity of H7 and its analogs is attributed to a direct and reversible interaction with the active center of protein kinases, acting competitively with respect to ATP (10). Under our experimental conditions, these three compounds did not affect the basal level of junB or PAI-1 mRNA, the basal rate of PAI-1 synthesis and incorporation into extracellular matrixes, or the phosphorylation of RB during middle to late G1 phase of the cell cycle. Thus, although the specificity of these protein kinase inhibitors is relatively broad, they did not cause a general perturbation of these cellular functions in short incubations. These inhibitors were also without effect on the binding of TGF-P to TGF-P receptors I and II and to the proteoglycan betaglycan. Therefore, one interpretation of these results is that H7, H8, and H9 inhibit early TGF-P responses by inhibiting a protein kinase(s) that mediates TGF-,B action. The involvement of an H7-sensitive protein kinase in multiple early gene responses to TGF-,B implies that TGF13-induced protein phosphorylation may modify, directly or indirectly, the activity of certain transcription factors. In agreement with this possibility, a recent study has shown that TGF-p1 induces the rapid phosphorylation of CREB and that this event could be coupled to increased junB expression (14). CREB is a transcription factor that is also phosphorylated by protein kinase A and calmodulin-dependent kinases in response to agents that elevate cyclic AMP or intracellular Ca2" levels, respectively (31, 34). CREB phosphorylation in response to TGF-1 does not appear to involve protein kinase A (14). It is of interest that the inhibition of RB protein phosphorylation by TGF-pi can be blocked by H7 and related compounds. Acting alone, H7 and its analogs do not prevent RB phosphorylation. These observations provide evidence that TGF-P controls the phosphorylation state of RB through a protein kinase that directly or indirectly antagonizes the action of the G1-specific RB kinase(s). The identity of the H7-sensitive protein kinase(s) that

mediates the TGF-P response is unknown. In vitro, H7 displays similar inhibitory activities towards protein kinases C, A, and G, whereas H8 and H9 display stronger inhibitory activities towards protein kinases A and G (10). The greater potency of H7 compared with H8 or H9 as inhibitors of the TGF-1 response might suggest the involvement of a protein kinase C. This possibility, however, is unlikely because cell responsiveness to TGF-P is resistant to the potent protein kinase C inhibitor, staurosporine. Furthermore, TGF-P responsiveness is retained in PMA-desensitized cells and, at least in the PAI-1 effect, is additive with that of PMA. A probable mechanism for transmembrane signalling by TGF-P and related factors is suggested by the recent cloning of an activin receptor (22). The deduced primary structure of this receptor suggests that it is a ligand-activated serine/ threonine kinase. Since the receptors for other members of the TGF-,B family might themselves constitute a family of structurally related proteins, it is conceivable that the activity affected by H7 and related compounds is a protein kinase activity intrinsic to the TGF-P receptor itself. However, our evidence based solely on the use of protein kinase inhibitors could obviously reflect the involvement of a kinase at any level in the pathway of TGF-P signal transduction. Another limitation of this approach is that H7 and its analogs might affect enzyme activities or cellular processes other than protein serine/threonine kinases. We know of no reports on such short-term side effects, but unlikely as these may seem, they remain a formal possibility. More definitive studies on this subject shall be possible once the TGF-P receptor structure is elucidated and the receptor protein becomes available for biochemical analysis.

ACKNOWLEDGMENTS This study was supported by National Institutes of Health grant CA 34610. M.O. is supported by Sankyo Co. Ltd., Tokyo, Japan.

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