Okadaic Acid Mimics Multiple Changes in Early Protein Phosphorylation and Gene Expression Induced by Tumor Necrosis Factor or Interleukin- 1”

Okadaic acid, a phosphatase inhibitor from a marine organism, mimics tumor necrosis factor/interleukin- 1 (TNF/IL-1) in inducing changes in early cellular protein phosphorylation. A total of -116 proteins exhibit significant and concordant changes in phosphorylation or dephosphorylation within 15 min in human fibroblasts activated by either okadaic acid, TNF, or IL-1. The fidelity of this mimicry by okadaic acid extends to the phosphorylation of the 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, epidermal growth factor receptor, and other cdc2-kinase substrates (c-abl, RB, and p53). The okadaic acid-induced pattern of protein phosphorylation is distinct from that observed in cells treated with phorbol 12-myristate 13-acetate or with ligands like epidermal growth factor, cyclic AMP agonists, bradykinin, or interferons. Like TNF, okadaic acid also induces the transcription of immediate early response genes like c-jun and Egr-1 as well as the interleukin-6 genes. The overall early effects of okadaic acid uniquely parallel those of TNF/ IL-1 and not those of other cytokines or ligands. Reg- ulation of protein phosphatase inhibition is discussed as a mechanism for TNF/IL- 1 signal transduction.

The control of the immune system is mediated through a complex interplay of lymphokines with different kinds of cells (1,2). TNF' and IL-1 are examples of lymphokines reported to be involved in the regulation of the host immune system (3-5). TNF and IL-1 are associated with disease states such as sepsis and autoimmune disorders and are described as mediators of the inflammatory response (4). Elevated levels of serum TNF in cancer, AIDS, and malaria are related to weight loss in these diseases (7). The association of these lymphokines with the disease state has prompted the application of TNF antibodies or inhibitors in the treatment of septic shock and the use of IL-1 antibodies in rheumatoid arthritis (8,9). It also prompted us to search for natural products which could mimic or antagonize TNF/IL-1 activity and/or signal transduction in primary human cells.
IL-1 was originally described as a comitogen of thymocytes and TNF was first reported to be an endotoxin-induced serum factor which causes tumor necrosis (10,ll). The two lymphokines have similar biological activities on cells even though they do not have homologous amino acid sequences or homology in their receptors (12)(13)(14)(15). Unlike the EGF, PDGF, and colony-stimulating factor-1 receptors which are known to have tyrosine kinase activity (16) and "serpentine receptors" that generate second messengers via GTP-binding proteins (17), little has been deduced of the mode of TNF or IL-1 signal transduction from their receptors. The involvement of GTP-binding proteins in TNF and IL-1 signalling processes was implied from several reports (18,19). Increases in second messenger concentrations of CAMP, inositol 1,4,5trisphosphate, and arachidonic acid have also been reported (20). The identification of several phosphorylated cellular substrates like the heat shock proteins, 1-plastin, stathmin, talin, and EGF receptor (EGFR) in TNF or IL-1 stimulated cells suggested the activation of serine/threonine kinase(s) (21,22). In this connection, both lymphokines have been reported to concordantly induce early changes in the phosphorylation of 63 cytosolic proteins and to activate multiple protein kinases including microtubule-associated protein-2 and eIF-4E kinases in human fibroblasts (23).
TNF or IL-1 activates a number of transcription factors including NF-kB, NF-IL6, multiple response factor, interferon response factor-1, interferon response factor-2, c-fos, cmyc, c-jun, and NF-GHa and induces the expression of several genes (24). Much has been described of TNF and IL-1 signalling effects at the transmembrane, cytosol, and gene expression levels, but the primary signalling pathway remains unresolved and in certain cases contradictory (21, 22,25). We present data to show that okadaic acid, a phosphatase inhibitor derived from a marine black sponge (26), induces a composite pattern of early phosphorylation changes and gene expression in primary human fibroblasts strikingly in common with those induced by TNF or IL-1. This mimicry of signalling effects introduces another dimension to the mechanism of TNF/IL-1 signal transduction in which the regulation of protein phosphatase activity is shown to play a role.

EXPERIMENTAL PROCEDURES
Cell Cultures and Reagents-Human diploid fibroblasts, FS-4, were gifts from Dr. J. Vilcek (New York University). They were grown in tissue culture flasks and maintained with minimal essential medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) hereon referred to as regular medium. Human WISH cells were purchased from the American Type Culture Collection. Recombinant human TNF and IL-1 with specific activities of 2 X lo7 units and 1 X 10' units/mg protein, respectively, and of purities of 299% were orthophosphate, cells were plated onto 90-mm plastic tissue culture dishes and incubated for 3 days for the cells to become confluent. Immediately before labeling, the media was removed and the cells were washed twice with 5 ml of phosphate-and serum-free media (150 mM NaCI, 5 mM MgC12, 5 mM KC1, 1.6 mM CaCl,, 0.5% glucose, 10 mM Tris, 0.1% bovine serum albumin, pH 7.4) before the addition of 1 mCi/ml of [32P]orthophosphate in 3 ml of the same media to each plate. The plates were incubated at 37 "C in a humidified C02 incubator for 2 h before the addition of the okadaic acid or other agonists at the concentrations and for the times indicated in the figures and table. The agonists were reconstituted in aqueous buffered solution or in dimethyl sulfoxide. Appropriate controls which were run with buffer or dimethyl sulfoxide in medium alone showed no detectable differences in the resulting autoradiographs. At the completion of labeling the media was removed, and the cells were rapidly washed twice with 5 ml of ice-cold phosphate-buffered saline before being solubilized in 200 pl of cytosol extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 p M Na3V04, 0.65% Nonidet P-40,2 mM leupeptin, 2 mM phenylmethylsulfonyl fluoride, pH 7.4). The lysate was collected and centrifuged for 5 min at 14,000 rpm. The pellet containing the nuclear fraction (including the cytoskeletal-membrane fractions) was separated from the cytosolic fraction. Both fractions were lyophilized.
Two-dimensional Gel Electrophoresis-The lyophilized fractions (each of -12 pg) were subjected to isoelectric focusing for 18,000 V/ h with pH 3-10 ampholytes using the Millipore Investigator twodimensional electrophoresis system, details of which are as previously described (23) (Millipore). Following this, the first-dimension gel was extruded and equilibrated in SDS buffer for 2 min before being loaded onto the second-dimension 12.5% SDS-polyacrylamide gel. The gel was fixed, dried, and "P-labeled polypeptides were located by autoradiography by using intensifying screens at -80 "C. For measurement of the PI profile, carbamylated protein standards (Pharmacia LKB Biotechnology Inc.) were co-run on duplicate gels and related to proteins of known PI on previously calibrated samples. Molecular weight marker proteins from Sigma were detected by Coomassie Blue staining and marked on the autoradiogram. 32P-Labeled 27 hsp complex prepared from human fibroblasts heat shocked at 43 "C for 30 min was also used as markers.
Quantitative Determination of Protein Phosphorylations-The amount of protein phosphorylation on autoradiographs derived from cytosolic membrane or antibody-precipitated preparations containing equal amounts of protein were analyzed by computerized densitometry using a Visage 2000 Image analysis system (BioImage Products, Ann Arbor, MI).
Preparation of RNA and Northern Analysis-Human fibroblasts (4 X lo6) were seeded in 175-cm2 tissue culture flasks and incubated at 37 "C for 6 days in regular medium. The cells were fed with regular medium on day 3. On day 6 the medium was replaced by minimal essential medium containing 0.25% fetal calf serum and incubated for another 48 h to maintain the cells in a quiescent state. Quiescent or regularly maintained cells were treated with a specific agonist for the times indicated (see Fig. 7). The cells were harvested and total cytoplasmic RNA isolated by a one-step guanidinium thiocyanate/ phenol/chloroform method. RNA was subjected to electrophoresis through 1.3% agarose gels containing 6.5% formaldehyde and then transblotted to Hybond-N membrane (Amersham, Buckinghamshire, UK). The blots were subjected to UV cross-linking and hybridized in a solution containing 0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, 1% bovine serum albumin, and 1 mM EDTA. Hybridization was carried out at 65 "C for 18 h with a 32P-labeled probe (final concentration of 2 x lo6 cpm/ml) generated by random priming on DNA templates. A BglII fragment of 1.6 kb corresponding to nucleotides 302-1958 of Egr-1 cDNA was used in the hybridization. The IL-6 cDNA probe was from the American Type Culture Collection (Rockville, MD) and the c-jun cDNA probe was from Dr. D. Carter of our institute. The membranes were exposed to x-ray films at -85 "C for 14 h to 7 days and rehybridized with &actin probe. The blots were autoradiographed and quantitated by the Visage 2000 Image system (BioImage Products, Ann Arbor, MI).

Changes in Early Phosphorylation of Cellular Proteins in Cells Treated with Okadaic Acid, TNF, or Other Agonkts-
The 32P-labeled protein spots are resolved and detected by autoradiography of the high definition two-dimensional gel electrophoresis of cytosolic extracts prepared from "P-labeled human fibroblasts. The radioactive spots (each representing one or more phosphoproteins) are analyzed by computerized densitometry. Each spot is arbitrarily assigned a number. In addition, the intensity of each resolved spot derived from extracts of cells previously treated with an agonist is expressed relative to a basal level of phosphorylation in the absence of agonist. These changes in the level of phosphorylation are matched against several internal standards during the computerized densitometry analysis. A typical example of the autoradiography of the two-dimensional gel analysis of the 32P-labeled phosphoproteins derived from the cytosolic or nuclear/cytoskeletal membrane fractions prepared from cells treated with either TNF (50 units/ml) or okadaic acid (200 nM) for 15 min is shown i n Fig. 1. The prominent changes to protein phosphorylation in the ligand activated cells are indicated (arrows). In the cytosolic fraction, okadaic acid induced the same changes (-95% identity) in early protein phosphorylation as TNF (Fig. 1, b and c). The phosphorylation pattern induced by IL-1 is not shown in Fig. 1 since the similarity between IL-1 and TNF has already been established (23). Okadaic acid also induced almost identical changes (as identified by computerized densitometry) in protein phosphorylation of the 46-50 proteins in the nuclear/cytoskeletal membrane fraction as did TNF treatment (Fig. 1, e and f ). A summary of the changes in cytosolic protein phosphorylation is listed in Table I, providing a composite pattern of changes of protein phosphorylation in these fractions derived from human fibroblasts activated for 15 min by either TNF, oka-    (Table I). TPA (an activator of protein kinase C) treatment of human fibroblasts produced a pattern of phosphorylation changes quite different from that produced by okadaic acid or TNF (Table I). TPA in combination with either okadaic acid or TNF produced a pattern of phosphorylation changes which represents the sum of complementary changes produced by each agonist (Table I). These results indicate that okadaic acid and TNF produce early changes in protein phosphorylation through a common pathway which is different from that of TPA-induced activation of protein kinase C. Similarly, the pattern of protein phosphorylation induced in cells treated by bradykinin, CAMP agonists, EGF, or IFN-a is different from that induced by okadaic acid or by TNF/IL-1 (data not shown). Furthermore, treatment of human fibroblasts by IFN-7 did not produce measurable changes in early protein phosphorylation. A number of the -66 proteins in the cytosol fraction and the 46-50 proteins in the nuclear/cytoskeletal membrane fraction which undergo the early phosphorylation changes have now been identified by either using marker proteins or by their molecular weights and isoelectric points. They include the 27 hsp complex (identified as 165, 172, and 176 in Table   I), stathmin (as S [27] in Fig. 1, b and c), nucleolin (as N [28] in Fig. 1, e and f), and myosin light chain (MLC in Fig. 1, e and f ). The phosphorylation of these proteins is concordantly enhanced in cells activated by TNF (Fig. 1, b and e), by okadaic acid (Fig. 1, c and f) or by IL-1 (not shown).
In a separate experiment using m7GTP-Sepharose to isolate the eIF-4E from okadaic acid-treated human fibroblasts by procedures previously reported (23) for TNF and IL-1, okadaic acid also enhanced the phosphorylation of this protein after 15 min by 2-3-fold (Fig. 2). Okadaic acid treatment of cells was extended to WISH cells and again produced almost identical changes in early protein phosphorylation as TNF (not shown), indicating that the mimicry of the early events of TNF signal transduction by okadaic acid is not confined to primary human fibroblasts.
It is important to note that though the TNF or IL-1 induced changes in protein phosphorylation shown in Fig. 1 and Table  I were measured 15 min after ligand treatment, these changes were detectable within 3 min and maximal at -15 min before returning to basal levels at 45-60 min. By then most of the changes in phosphorylation induced by TNF or IL-1 are no longer measurable.
Dose-response and Time Course of Olzadaic Acid-induced Changes in the Phosphorylation of the 27 hsp Complex-The 27 hsp complex is most sensitive to phosphorylation changes induced by okadaic acid and TNF (Fig. 1). The concentrationdependent effect of okadaic acid on changes in phosphorylation of the 27 hsp complex was measured and compared with those in cells treated with 50 units/ml of TNF (Fig. 3). It is apparent from this comparison that treatment of cells with 50 units/ml of TNF for 15 min produces about the same increase in the phosphorylation of the 27 hsp complex as treatment of cells with between 160-320 nM okadaic acid for 15 min (Fig. 3, c-e).
A  Protein Phosphatase in TNFIIL-1 Signal Transduction phorylation (29,30). Okadaic acid was added to human fibroblasts to see if it can also mimic this effect of TNF and IL-1. '"P-Labeled human fibroblasts were treated with ligand (TNF, IL-1, EGF, PDGF, or okadaic acid) for 15 min and extracted for analysis. The extracts were immunoprecipitated with anti-EGFR antibodies. The immunoprecipitated EGFR derived from variously treated fibroblasts was analyzed by SDS-PAGE and autoradiography. Okadaic acid significantly stimulates the phosphorylation of the EGFR to an even greater extent than does TNF, IL-1, EGF, or PDGF (Fig. 5).
Phosphorylation of cdc2 Kinase Substrates in Cells Treated with IL-1, TNF, or Okadaic Acid-It is noted from Fig. 1 (e and f ) that the phosphorylation of myosin light chain and nucleolin are enhanced by TNF and okadaic acid treatment. Both proteins are known to be substrates of cdc2 kinase. In this connection, a question was raised as to whether other cdc2 kinase substrates which are known to be phosphorylated by okadaic acid treatment (31,32) are also affected in TNFtreated human fibroblasts. Three substrates of cdc2 kinase, namely c-abl, RB, and p53, were tested. Like okadaic acid, TNF induced the phosphorylation of all three cdc2 kinase substrates in human fibroblasts. Okadaic acid or TNF enhanced the phosphorylation of c-abl, RB, and p53 (Fig. 6). The results also show that the RB proteins are found in two forms, one having slightly higher molecular mass (115 kDa) than the other (110 kDa). The 115-kDa protein is likely the hyperphosphorylated RB protein, whereas the 110-kDa protein represents the hypophosphorylated form (33). Like okadaic acid treatment of cells, TNF increases the ratio of the hyperphosphorylated RB protein to the hypophosphorylated form of RB (Fig. 6). Similarly the p53 protein is present in two very closely associated forms (34), the slightly higher molecular mass species being more heavily phosphorylated than the slightly lower molecular mass species. Like TNFtreated cells, okadaic acid-treated fibroblasts showed 3-to 25-fold higher amounts of the hyperphosphorylated RB and p53 protein than the lesser phosphorylated forms of these proteins.
Induction of Egr-1, c-jun, and ZL-6 Genes by TNF and Okadaic Acid-Having shown the early changes in protein phosphorylation in the cytosol and nuclear/cytoskeletal membrane fractions of okadaic acid-, TNF-, and IL-1-treated fibroblasts, we investigated the effects of okadaic acid on gene expression. TNF is known to induce eight genes in primary human fibroblasts (35). Recently we observed the induction of an immediate early response gene, the Egr-1 gene, in primary human fibroblasts in response to TNF. The induction of expression of three TNF/IL-1-inducible genes by okadaic acid was preliminarily examined in primary human fibroblasts. The three genes selected were Egr-1, c-jun (36) and IL-6 (37). Like TNF, okadaic acid effectively induced all these  ( a ) or OA (500 nM) ( b ) for 0,30,60 min after which total cellular RNA was extracted. Blots of RNA from these cells were hybridized to radiolabeled (lo6 cpm/ml) Egr-1 cDNA probe. c, the blot in b was stripped and reprobed with c-jun cDNA. Note: Egr-1 mRNA probe was not completely stripped from the blot. The autoradiograph of the blot is presented. d, regularly maintained cells were incubated either with no additions (control, 0 time) or with OA for 1, 3, 5, 7 h, and Northern analysis was carried out with an IL-6 cDNA probe (ATCC, Rockville, MD). genes. Egr-1 was rapidly induced within 30 min and c-jun within 60 min of okadaic acid treatment, whereas ZL-6 was induced much later at 7 h after okadaic acid treatment (Fig.  7). We have no explanation concerning the late induction of ZL-6 by okadaic acid except to note that similar late induction of c-fos by okadaic acid was recently reported as well (43).

DISCUSSION
In this study, we found that okadaic acid induces changes in early protein phosphorylation in primary human fibroblasts similar to that induced by TNF or IL-1 ( Fig. 1 and Table I). Out of the 116 proteins affected by treatment of cells with okadaic acid or TNF or IL-1,60% of the changes were in the cytosolic fraction and 40% in the nuclear and cytoskeletal/ membrane fractions (Fig. 1). A number of these phosphoproteins were identified, including the EGFR, 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, and cdckinase substrates such as c-abl, RB, and P53 proteins (Figs.  1, 2, 5, and 6). Like TNF, okadaic acid also induces the transcription of Egr-I, c-jun, and IL-6 genes in human fibroblasts (Fig. 7). However, unlike TNF or IL-1 the protein phosphorylation induced by okadaic acid increased proportionally with the time of treatment (Fig. 4). No subsequent decrease was observed during the first 2 h of treatment. Longer treatment (30 min or more) resulted in the hyperphosphorylation of proteins. In this respect, the changes induced by okadaic acid differ from those observed upon TNF/IL-1 treatment which produces transient increases in cellular protein phosphorylation. An explanation for this difference is that IL-1 and TNF receptors are known to be internalized upon ligand activation and hence their effects can be down-regulated with time (23). On the other hand, okadaic acid accumulates in the treated cells and continues to exert its effect on the phosphorylation of the target proteins resulting in the hyperphosphorylation of these target proteins in the cell. Other than this, the fidelity of mimicry of the early events of TNF or IL-1 action by okadaic acid is maintained from the levels of cytosolic and nuclear protein phosphorylation to that of gene induction ( Fig. 1 and Fig. 7). In comparison with other ligands [namely TPA (Table I), CAMP agonists, EGF, bradykinin, or ZFN (not shown)J, TNF, ZL-1, or okadaic acid induce a distinct pattern of cellular protein phosphorylation. In addition even though okadaic acid can mimic a few aspects of EGF action, such as the transmodulation of the EGFR phosphorylation (Fig. 5), it is evident from the comparison of the early changes in protein phosphoryation and gene induction that overall okadaic acid effects are uniquely parallel to those of TNF/IL-1 and not to those of other cytokines or ligands tested.
The striking similarity of changes in the phosphorylation of cellular proteins induced by okadaic acid and TNF reported herein indicates that when human fibroblasts are treated with either of the agonists, similar signal transduction pathways are initiated. In this regard, the inhibitory effect of okadaic acid on protein phosphatase-2A and to a lesser extent on protein phosphatase-1 (38) provides clues to a possible mechanism of its mimicry of TNF/IL-1 actions as well as to the mechanism of TNF/IL-1 signalling itself. A number of explanations are discussed. It is possible that the phosphorylation state of the substrates of TNF/IL-1-activated protein kinase(s) is tightly regulated by an okadaic acid-sensitive phosphatase(s). The inactivation of the phosphatase(s) by okadaic acid would alter the balance of the opposing phosphorylation/ dephosphorylation in favor of net phosphorylation. Another explanation is that TNF/IL-1 treatment leads to the inactivation of an okadaic acid-sensitive protein phosphatase(s). In this scenario the inhibition of dephosphorylation will, as above, result in an overall increase in the phosphorylation of the cellular protein substrates by the opposing protein kinase. This explanation would account for the relatively large number of proteins showing net increases in phosphorylation after cells are treated with TNF/IL-1 or okadaic acid. It is also possible that okadaic acid treatment can indirectly activate the same protein kinases that TNF/IL-1 treatment activates. The indirect activation of these kinases can occur via the inhibition of a kinase suppressor, a suppressor which is active in the dephosphorylated form. In this connection, okadaic acid has been shown to possibly activate cdc2 and microtubule-associated protein-2 kinase through this mechanism (39-41). The inactivation of a protein phosphatase as a signalling mechanism is unprecedented. There are several examples of specific protein phosphorylations that are regulated at the level of protein kinase(s) and phosphatase(s1 (42). The present finding of the mimicry of TNF/IL-1 by okadaic acid introduces the concept of protein phosphatase regulation as one important mechanism by which the signal transduction of TNF/IL-1 is mediated.