Transforming Growth Factor β1 Rescues Serum Deprivation-induced Apoptosis via the Mitogen-activated Protein Kinase (MAPK) Pathway in Macrophages*

Cell death and cell survival are central components of normal development and pathologic states. Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine that regulates both cell growth and cell death. To better understand the molecular mechanisms that control cell death or survival, we investigated the role of TGF-β1 in the apoptotic process by dominant-negative inhibition of both TGF-β1 and mitogen-activated protein kinase (MAPK) signaling pathways. Murine macrophages (RAW 264.7) undergo apoptosis following serum deprivation, as determined by DNA laddering assay. However, apoptosis is prevented in serum-deprived macrophages by the presence of exogenous TGF-β1. Using stably transfected RAW 264.7 cells with the kinase-deleted dominant-negative mutant of TβR-II (TβR-IIM) cDNA, we demonstrate that this protective effect by TGF-β1 is completely abrogated. To determine the downstream signaling pathways, we examined TGF-β1 effects on the MAPK pathway. We show that TGF-β1 induces the extracellular signal-regulated kinase (ERK) activity in a time-dependent manner up to 4 h after stimulation. Furthermore, TGF-β1 does not rescue serum deprivation-induced apoptosis in RAW 264.7 cells transfected with a dominant-negative mutant MAPK (ERK2) cDNA or in wild type RAW 264.7 cells in the presence of the MAPK kinase (MEK1) inhibitor. Taken together, our data demonstrate for the first time that TGF-β1 is an inhibitor of apoptosis in cultured macrophages and may serve as a cell survival factor via TβR-II-mediated signaling and downstream intracellular MAPK signaling pathway.

Apoptosis, the process of programmed cell death, is an integral part of normal embryonic development, inflammatory response, and tumorigenesis (1). It is a highly regulated series of well coordinated events characterized by distinctive morpho-logic and biochemical changes involving nuclear and chromatin condensation, cell membrane blebbing, and loss of cellular integrity forming distinct apoptotic bodies, as well as endonuclease activity resulting in DNA fragmentation and ultimately cell death (2). Regulatory mechanisms controlling cell death is as fundamental as those regulating cell growth in achieving the homeostatic balance between cell survival and cell death and involve a complex interplay of specific regulatory genes in signaling cells to either live or die.
Transforming growth factor ␤ 1 (TGF-␤ 1 ) 1 is a 25-kDa polypeptide, belonging to a superfamily of multifunctional cytokines, that regulates cellular growth and differentiation and extracellular matrix production (3). Moreover, TGF-␤ 1 has been shown to be a potent modulator of apoptosis in a variety of cell types, including epithelial cells, hepatocytes, hematopoietic cells, and lymphocytes, which undergo programmed cell death in response to TGF-␤ 1 (4 -7). We have previously reported the induction of apoptosis by TGF-␤ 1 in endothelial cells (8). However, more recent studies suggest that TGF-␤ 1 also possesses the ability to inhibit apoptosis, further affirming the multifunctional nature of this cytokine (9).
TGF-␤ 1 elicits multiple biological responses by interaction with two transmembrane receptor serine/threonine kinases known as TGF-␤ type I receptor (T␤R-I) and TGF-␤ type II receptor (T␤R-II) (3). T␤R-II is a constitutively active kinase, which binds TGF-␤ 1 directly and recruits T␤R-I to form a "heteromeric" complex, and the signaling cascade is initiated upon transphosphorylation of the GS domain of T␤R-I by T␤R-II (10). T␤R-I alone does not exhibit significant binding of TGF-␤ 1 ligand when assessed by cross-linking analysis, and T␤R-II is unable to signal without T␤R-I (10). Thus, T␤R-II is required for initial ligand binding and phosphorylation of T␤R-I to initiate the signaling cascade. We have previously reported the critical role of T␤R-II in the TGF-␤ 1 signaling pathway to induce apoptosis in endothelial cells (8). Interference with T␤R-II-mediated signal transduction by a dominantnegative mutant of T␤R-II blocked TGF-␤ 1 -induced endothelial cell apoptosis and associated capillary morphogenesis in vitro (8).
Although molecular cloning of the TGF-␤ receptors have furthered our understanding of the mechanism of TGF-␤ 1 signaling, the downstream signaling pathways activated after the initial receptor interaction with ligand to mediate multiple TGF-␤ 1 responses remain poorly understood. Recent studies support the involvement of the mitogen-activated protein kinase (MAPK) pathways in TGF-␤ 1 signaling (11)(12)(13)(14). Moreover, activation of the MAPK-dependent pathways has been implicated in the process of apoptosis (15,16). Members of the MAPK family, like the TGF-␤ receptors, are structurally related serine/threonine kinases that are actively involved in cellular events such as growth, differentiation, and cellular responses to environmental stress (17,18). There are three groups of the MAPK family members identified to date: the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), also known as p44 and p42 MAPKs, respectively; the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/ SAPK); and the p38 (18,19). The signal transduction cascades involved in the activation of MAPKs require a well coordinated series of three protein kinase reactions, propagating the phosphorylation and the activation of the next kinase in their respective pathways. The MAPKs require dual phosphorylation at the threonine and tyrosine sites by MAPK kinases, the MEKs and MKKs that are specific for ERK, JNK, and p38, which are in turn activated by MAPK kinase kinases (MKKKs) via serine/threonine phosphorylation (19,20). The MAPK cascades display evolutionary conservation and are implicated to play essential roles in the regulation of cell growth, differentiation, and apoptosis.
To better understand the molecular mechanism controlling cell death or survival, we investigated the role of TGF-␤ 1 in the apoptotic process by dominant-negative inhibition of both TGF-␤ 1 and MAPK signaling pathways. In this study, we utilized serum withdrawal or deprivation to induce apoptosis by decreased availability of cell survival factors. We show that serum deprivation induces apoptosis in murine macrophages (RAW 264.7) and that TGF-␤ 1 is able to prevent serum-deprived macrophages from undergoing apoptosis. This "rescue" is inhibited in cells transfected with a dominant-negative mutant of T␤R-II (T␤R-II M ), suggesting the critical role of T␤R-II in TGF-␤ 1 signaling to prevent serum deprivation-induced apoptosis. Furthermore, we demonstrate that TGF-␤ 1 rapidly induces ERK1/ERK2 MAPK activity. TGF-␤ 1 fails to rescue RAW 264.7 cells from serum deprivation-induced apoptosis upon stable transfection with a dominant-negative mutant MAPK (ERK2) cDNA or in the presence of the MEK1 inhibitor. Taken together, our data suggest that TGF-␤ 1 rescues macrophages from serum deprivation-induced apoptosis via T␤R-IImediated signaling and downstream intracellular MAPK signaling pathway.
Constructs-A truncated T␤R-II construct (T␤R-II M ), lacking the serine/threonine kinase domain, but containing the full transmembrane spanning and extracellular domains, was generated by polymerase chain reaction (PCR) using a rat T␤R-II cDNA as the template, as described previously (8). Primer sequences were as follows: sense primer 5Ј-GTTAAGGCTAGCGACGGGGGCTGCCATG-3Ј; antisense primer 5Ј-GGCGGTCGACTAGACACGGTAACAGTAGAAG-3Ј. These contained the sequences for the restriction enzymes NheI and SalI, respectively (underlined), for directional cloning, and a stop codon in the antisense primer. The PCR-amplified product was cloned into the pMAMneo (CLONTECH), a glucocorticoid-inducible mammalian expression vector, containing a neomycin-resistant gene. Correct directionality and in-frame sequences of the PCR product ligated in pMAMneo were verified by restriction mapping with EcoRI, BamHI, and HindIII and sequencing by the dideoxy chain termination technique using Sequenase 2.0 (United States Biochemical Corp.). The MAPK-WT (wild type ERK2) and the MAPK-TA (dominant-negative mutant of ERK2) constructs used in this study were provided by Dr. Andrew Larner (21).
Cell Culture and Transfection-The murine peritoneal macrophage cell line, RAW 264.7, was obtained from ATCC (Rockville, MD). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% FBS (HyClone) and gentamicin (50 g/ml) in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. To generate clones that stably expressed T␤R-II M , RAW 264.7 cells were transfected using Lipofectin (Life Technologies, Inc.) as follows. Cells grown to approximately 50% confluency on 100-mm dishes (Falcon) were incubated with 10 g of DNA (T␤R-II M ) and 50 l of Lipofectin suspension in DMEM at 37°C in 5% CO 2 . After a 5-h incubation, medium containing 20% FBS in DMEM was added to make a final concentration of 10% FBS and incubated further for 48 h. Then the DNA/Lipofectin-containing medium was changed to 10% FBS in DMEM (no antibiotics) and incubated for another 24 h. To select for transfectants, cells were treated up to 800 g/ml G418 (Life Technologies, Inc.) in DMEM containing 10% FBS, and the medium was changed every 2-3 days. G418-resistant colonies emerged at approximately 10 days after transfection and were subcloned using ring cylinders, expanded, and maintained in DMEM containing 10% FBS, Geneticin (200 g/ml), and gentamicin (50 g/ml). Two independent, stably transfected clones expressing the T␤R-II M , named 10-2 and 10-3, were expanded. Confirmation of mRNA expression was obtained by reverse transcription-polymerase chain reaction using primer pairs that contain speciesspecific sequences that recognize only the transfected T␤R-II M construct and not the endogenous wild-type T␤R-II.
To generate clones that stably expressed the MAPK-WT or the MAPK-TA, the corresponding constructs were co-transfected with pcDNA3 (Invitrogen), a mammalian expression vector containing a neomycin-resistant gene, using Lipofectin, as described above. The stable transfectants were also selected in medium containing 800 g/ml G418 and then subcloned and maintained in 200 g/ml G418. Confirmation of mRNA expression was obtained by reverse transcription-PCR.
Induction of Apoptosis/Genomic DNA Isolation and Analysis-To induce apoptosis, cells grown on 100-mm dishes (Falcon) to 90% confluency were placed in DMEM containing 0.5% FBS for 24 h. In experiments involving treatment with cytokines, the cells were incubated in the absence or presence of exogenous TGF-␤ 1 (1 ng/ml-100 ng/ml) or TGF-␣ (1 ng/ml-100 ng/ml) at 37°C for 24 h. In experiments with MEK1 inhibitor, PD098059, cells were incubated in the absence or presence of 30 M PD098059 for 24 h. The concentration of 30 M was chosen as it is the optimal concentration inhibiting ERK without imparting cellular toxicity in these cells. For experiments involving exposure of RAW 264.7 cells to hyperoxia, the cells were placed in a tightly sealed modular chamber (Billup-Rothberg, Del Mar, CA) with 5% CO 2 and 95% O 2 at 37°C. Control cells were maintained in 5% CO 2 and 95% air at 37°C.
Genomic DNA isolation was performed using the Puregene kit (Gentra Systems, Inc.) according to the manufacturer's directions. Briefly, cells were lysed directly on the plate after medium removal with lysis buffer followed by a 1-h incubation with RNase A. The cell lysates were precipitated for proteins and spun at 2000 ϫ g for 15 min. Then, isopropyl alcohol was added to the supernatant to precipitate the DNA. After an alcohol wash, the DNA was hydrated and quantified, and 20 g was analyzed on 1.5% agarose gel electrophoresis. The T␤R-II M stable transfectants were preincubated in the presence or absence of 1 M dexamethasone for 24 h prior to serum deprivation and treatment with exogenous TGF-␤1. The MAPK-WT and MAPK-TA stable transfectants were also subjected to serum withdrawal and TGF-␤ 1 as described above. Each of the experiments was repeated at least three times.
Cell Survival Assay-Determination of cell viability was done by trypan blue exclusion assay. Cells grown on 12-well plates to 90% confluency were induced to undergo apoptosis as described above, and at the indicated time periods, cells in each of the wells were collected, centrifuged, and resuspended in 0.5 ml of DMEM. Then aliquots of 0.1 ml were incubated with trypan blue dye (Life Technologies, Inc.) for 5 min followed by cell counting by hemocytometer. Both live (unstained) and dead (blue) cells were counted from the same randomly selected fields. The results were expressed as percentages of surviving cells that did not take up the trypan blue dye in the total cell population. The experiments were performed in triplicate and repeated two times.
MAPK (ERK1 and ERK2) Activity Assays-Kinase assays were performed as described by Marais et al. (22) with minor modifications. Briefly, cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride and sonicated. Protein concentrations were determined as described for Western analysis. Total protein (200 g) samples were incubated with phosphospecific p44/42 MAPK rabbit polyclonal antibody (1:50) overnight on a rocker at 4°C. For a positive control, 20 ng of active MAPK (ERK2) was incubated with control cell extract. Protein A-Sepharose beads (Amersham Pharmacia Biotech) were then added to immunoprecipitate the activated MAPK complex. The immunoprecipitate pellets were incubated with 1 g of Elk-1 fusion protein in the presence of 100 M ATP and a kinase buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM ␤-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , and 10 mM MgCl 2 . The reaction was terminated with SDS loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% w/v bromphenol blue). The samples were analyzed on 12% SDS-PAGE and electroblotted as described for Western blot. ERK activity was assayed by detection of phosphorylated Elk-1 using a phosphospecific Elk-1 rabbit polyclonal antibody (1:1000). After overnight incubation with the primary antibody at 4°C, the membrane was incubated for 1 h with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) at room temperature with gentle rocking. The proteins were subsequently detected using LumiGLO (New England Biolabs) and exposed to x-ray film. All of the assays were repeated three times.
Statistical Analysis-Statistical significance of the experimental data for the cell survival assays by trypan blue exclusion was determined by analysis of variance or the Student's t test for paired data, as appropriate. p values Ͻ 0.05 were considered significant. Data are presented as means Ϯ S.E. of triplicate determinations.

Serum Deprivation Induces Apoptosis in RAW 264.7
Cells-We first determined whether RAW 264.7 cells underwent apoptosis following withdrawal of serum. Genomic DNA isolated from RAW 264.7 cells were assessed for the presence of DNA fragmentation by a "ladder" pattern on agarose gel electrophoresis, indicative of internucleosomal cleavage, a hallmark of apoptosis. The induction of genomic DNA fragmentation was observed in RAW 264.7 cells after 24 h of serum deprivation (Fig. 1).
TGF-␤ 1 Rescues Serum-deprived RAW 264.7 Cells from Apoptosis-Given that previous studies have implicated the role of TGF-␤ 1 as a modulator of apoptosis, we examined the effects of TGF-␤ 1 on serum deprivation-induced apoptosis in RAW 264.7 cells. As shown in Fig. 2A, genomic DNA fragmentation was not observed in RAW 264.7 cells upon serum deprivation in the presence of exogenous TGF-␤ 1 (lanes 3-5, 1, 10, and 100 ng/ml, respectively). This inhibition of DNA fragmentation by TGF-␤ 1 was associated with increased cell survival, as shown in Fig. 3. Treatment with exogenous TGF-␤ 1 (10 ng/ml) resulted in increased cell survival of 93 Ϯ 2% compared with 78 Ϯ 3% after serum deprivation for 24 h (p Ͻ 0.01, Student's t test, n ϭ 3) and 88 Ϯ 4% compared with 49 Ϯ 2% cell survival after serum deprivation for 48 h (p Ͻ 0.005, Student's t test, n ϭ 3). The increased cell survival with TGF-␤ 1 treatment remained significant up to 96 h following serum deprivation. TGF-␣, an analog of epidermal growth factor, chemically distinct from TGF-␤ 1 and acting through a tyrosine kinase receptor system, failed to prevent DNA fragmentation in serum-deprived RAW 264.7 cells (data not shown). Furthermore, TGF-␤ 1 did not rescue the apoptotic process elicited by other stimuli such as oxidative stress (Fig. 2B), indicating specificity of TGF-␤ 1 -mediated rescue from serum deprivation-induced apoptosis in RAW 264.7 cells.
TGF-␤ 1 Rescues Serum Deprivation-induced Apoptosis via T␤R-II Signaling Pathway-To determine whether the ability of TGF-␤ 1 to rescue RAW 264.7 cells from serum deprivationinduced apoptosis was mediated by T␤R-II, we first generated stably transfected cells overexpressing a dominant-negative mutant of T␤R-II (T␤R-II M ). Given that TGF-␤ 1 signal transduction requires heterodimerization of T␤R-II and T␤R-I and transphosphorylation of T␤R-I by T␤R-II, the truncated receptor T␤R-II M , which is membrane-anchored but lacks the cytoplasmic serine/threonine kinase domain, competes for binding to T␤R-I, hence acting in a dominant-negative fashion to inhibit TGF-␤ 1 signaling (8, 23). As predicted, complete inhibition of TGF-␤ 1 rescue from serum deprivation-induced apoptosis was observed in cells from two independent clones (10-2 and 10-3) expressing the truncated receptors, T␤R-II M (Fig. 4). This occurred both with and without dexamethasone pretreatment. Although the T␤R-II M construct was under a glucocorticoidregulated promoter, "leakage" of promoter activity occurs during uninduced conditions and has been previously observed by us and other investigators (8,24).
TGF-␤ 1 Activates MAPK (ERK1 and ERK2) in RAW 264.7 Cells-Previous studies have suggested that TGF-␤ 1 exerts its biological effects via the MAPK signaling pathway in several cell culture systems (11)(12)(13)(14). We first determined the levels of ERK1 and ERK2 protein expression in RAW 264.7 cells treated with exogenous TGF-␤ 1 (10 ng/ml) by Western analyses, using phosphospecific p44/42 MAPK and p44/42 MAPK antibodies. The phosphospecific p44/42 MAPK antibodies detect specifically the phosphorylated forms of ERK1/ERK2, whereas the p44/42 MAPK antibodies detect total (phosphorylation-state independent) ERK1/ERK2 proteins. As shown in Fig. 5A, increases in phosphorylation of ERK1 and ERK2 proteins were observed in cells, as early as 15 min after stimulation with exogenous TGF-␤ 1 . There were no appreciable increases in the activation of JNK or p38 within the same time periods of TGF-␤ 1 treatment (Fig. 5B).
We next examined whether this induction of ERK1 and ERK2 by TGF-␤ 1 was associated with an increase in MAPK activity using an immunocomplex kinase assay. Lysates from RAW 264.7 cells incubated in the presence or absence or exogenous TGF-␤ 1 (10 ng/ml) were subjected to immunoprecipitation using phosphospecific p44/42 MAPK antibodies. The resulting active ERK1/ERK2 immunoprecipitate was then allowed to phosphorylate Elk-1 fusion protein, and ERK activity was assayed by the detection of phosphorylated Elk-1 by Western blot analysis. Exogenous TGF-␤ 1 (10 ng/ml) induced the increase of the phosphorylated form of Elk-1 (Fig. 5C). Although there was some endogenous activity in the control untreated cells, TGF-␤ 1 induced ERK activity within 15 min of TGF-␤ 1 treatment, with marked ERK activity up to 4 h of TGF-␤ 1 treatment.
Inhibition of Serum Deprivation-induced Apoptosis by TGF-␤ 1 Involves the MAPK Pathway-Given that TGF-␤ 1 activates the MAPK (ERK) pathway in RAW 264.7 cells, we next examined whether the MAPK signaling pathway mediates the TGF-␤ 1 rescue of RAW 264.7 cells from serum deprivationinduced apoptosis. Our first strategy was to inhibit the MAPK pathway by genetic blockade utilizing a dominant-negative mutant of ERK MAPK (MAPK-TA). Fig. 6A demonstrates that TGF-␤ 1 rescues serum deprivation-induced apoptosis in cells transfected with MAPK-WT, as was previously observed in wild-type RAW 264.7 cells. However, in RAW 264.7 cells that have been transfected with a dominant-negative mutant MAPK-TA, genomic DNA fragmentation was observed both in the presence of serum and upon serum deprivation, and treatment with exogenous TGF-␤1 failed to prevent apoptosis (Fig.  6B). This suggests that the rescue effect of TGF-␤ 1 is in part mediated by the MAPK signaling pathway.
Inhibition of MEK1 Prevents the Anti-apoptotic Effects of TGF-␤ 1 in Serum-deprived RAW 264.7 Cells-To further confirm the role of the ERK MAPK pathway in TGF-␤ 1 rescue of serum deprivation-induced apoptosis, we next utilized a selective inhibitor of MEK1 (upstream of ERK). In wild-type RAW 264.7 cells treated with 30 M of the MEK1 inhibitor PD098059 for 24 h, DNA fragmentation was observed both in the presence of serum (Fig. 7A, lanes 3 and 4) and upon serum deprivation (Fig. 7B, lanes 3 and 4), and treatment with exogenous TGF-␤ 1 failed to prevent apoptosis. Taken together, these results suggest that inhibiting MEK1 disrupts the signaling process of exogenous TGF-␤ 1 by preventing the activation of the MAPK pathway under conditions of serum deprivation, thus disrupting the initiation of apoptotic rescue. DISCUSSION It is now well accepted that the process of programmed cell death, or apoptosis, serves as a critical force in the development and homeostasis of multicellular organisms and is carefully regulated by diverse signals that influence the decision of a cell between life and death. These signals may act to either promote or inhibit apoptosis, and the same signal may potentially have opposing effects on different cell types. One such signaling molecule that may possess both pro-apoptotic and anti-apoptotic activities is the multifunctional cytokine TGF-␤ 1 , both a potent stimulator and an inhibitor of cell proliferation. In the present study, we examined the role of TGF-␤ 1 in modulating apoptosis in cultured macrophages (RAW 264.7 cells). In order to induce apoptosis, serum was withdrawn. Serum deprivation has been shown to provoke apoptosis in a variety of cells including fibroblasts and endothelial cells, as a result of decreased availability of cell survival factors (8,25). We observed that macrophages also undergo apoptosis upon serum withdrawal or deprivation, as determined by detection of the characteristic genomic DNA laddering (Fig. 1). Remarkably, the presence of exogenous TGF-␤ 1 prevented the macrophages from undergoing apoptosis upon serum withdrawal ( Fig. 2A). This inhibition of DNA laddering by TGF-␤ 1 was also associated with increased cell survival (Fig. 3). The anti-apoptotic activity of TGF-␤ 1 was further confirmed by its inability to rescue cells from serum deprivation-induced apoptosis when its signaling receptors are blocked by a kinase-deleted dominantnegative mutant of T␤R-II (T␤R-II M ). Since signal transduction requires heterodimerization of T␤R-II and T␤R-I, the mutant receptor competes for binding to wild-type T␤R-I, hence acting in a dominant-negative fashion (3,8,10). In stably transfected RAW 264.7 cells expressing the T␤R-II M , apoptosis occurred with serum deprivation, both in the presence or absence of exogenous TGF-␤ 1 (Fig. 4), indicating that the anti-apoptotic effect by TGF-␤ 1 is mediated by T␤R-II kinase.
Although TGF-␤ 1 has been shown in a number of systems to be a potent inducer of apoptosis, anti-apoptotic actions of TGF-␤ 1 are less well known. Sachsenmeier et al. reported "protective" effects of TGF-␤ 1 by inhibiting suspension-induced apoptosis in human keratinocytes following loss of adhesion (9). Treatment of keratinocytes with TGF-␤ 1 attenuated suspension-induced DNA fragmentation. Moreover, inhibition of endogenous TGF-␤ 1 by neutralizing antibody to TGF-␤ 1 increased DNA fragmentation following suspension. Thus, TGF-␤ 1 clearly possesses the ability to exert both pro-apoptotic and anti-apoptotic effects in different cell systems, and the differential cellular responses likely are necessary for proper homeostasis of multicellular organisms. With the findings that TGF-␤ 1 can promote cell survival in certain cell types, we were interested in exploring the potential downstream intracellular pathways responsible for these protective effects of TGF-␤ 1 in macrophages.
Evidences that TGF-␤ 1 is capable of activating MAPK-dependent pathways in mammalian cells have been reported. For instance, rapid activation of ERK1 by TGF-␤ 1 has been demonstrated in intestinal epithelial cells and is associated with growth inhibitory effects of TGF-␤ 1 (12). In other cell types, including HepG2, CHO, and MDCK cell lines, TGF-␤ 1 has been shown to activate JNK/SAPK, and dominant-negative forms of various components of the JNK/SAPK pathway abolished TGF-␤ signaling (13). We examined whether TGF-␤ 1 is capable of activating MAPK in macrophages and whether TGF-␤ 1 signals rescue from serum deprivation-induced apoptosis via the MAPK-dependent pathway. ERK activity was assayed by two methods. First, increased phosphorylation of ERK1/ERK2 was determined by Western analyses using phosphospecific p44/42 MAPK antibodies that detect only the tyrosine 204-phosphorylated forms of ERK1/ERK2. Next, ERK activity was determined by in vitro kinase assay. Following immunoprecipitation with p44/42 MAPK antibodies to select for the activated (phosphorylated) MAPK, detection of in vitro phosphorylation of a known substrate, Elk-1, was determined using phosphospecific antibodies that detect only the serine 383-phosphorylated Elk-1. Our results show that ERK1/ERK2 was activated within 15 min of stimulation with exogenous TGF-␤ 1 in cultured RAW 264.7 cells, and this activation was sustained up to 4 h (Fig.  5C). Accordingly, the sharp increase in phosphorylation of Elk-1 is observed parallel with increased phosphorylated forms of ERK1/ERK2 (Fig. 5A). In contrast, we observed that TGF-␤ 1 failed to activate JNK/SAPK or p38 within this same time period, indicating that TGF-␤ 1 is capable of rapidly activating only the ERK pathway, but not the JNK/SAPK or p38 pathways, in RAW 264.7 macrophages (Fig. 5B).
The ERK pathway is the prototypical MAPK pathway induced by epidermal growth factor stimulation and implicated in the regulation processes of cellular proliferation and differentiation (18,26). Evidence for its potential importance in the modulation of apoptosis has been provided by studies in cardiac myocytes. Cardiotrophin 1 (CT-1), a member of the interleukin 6 family of cytokines, is a potent cardiac survival factor capable of inhibiting apoptosis in cardiac myocytes via the activation of an anti-apoptotic signaling pathway that requires MEKs (MAPK/ERK kinases) (27). We determined whether the ERK pathway was involved in the apoptotic rescue of macrophages by TGF-␤ 1 , using two independent approaches to block the ERK signaling pathway. Our transfection studies with the dominant-negative mutant MAPK (ERK2) in RAW 264.7 cells resulted in the blockade of TGF-␤ 1 anti-apoptotic effects (Fig.  6B). To further support these findings, we utilized an MEK1specific inhibitor, PD098059, which blocks MEK1 activation by Raf, thus preventing downstream activation of ERK1/ERK2, but does not inhibit JNK/SAPK or p38 protein kinase activation. In addition, the PD098059 has been shown to have little effect on other kinases, including cAMP-dependent kinase, protein kinase C, and other serine and threonine kinases (28 -30). In our studies, PD098059 effectively prevented the anti-apoptotic effects of TGF-␤ 1 in serum-deprived macrophages and provides further evidence for the requirement of the ERK pathway in the survival function of TGF-␤ 1 (Fig. 7B).
Interestingly, studies supporting our current findings that MAPK-dependent pathways are responsible for promoting the survival effects of TGF-␤ 1 have been documented for other cytokines in neuronal cells and cardiac myocytes. Nerve growth factor promotes the survival of neuronal (PC-12) cells via activation of the ERK pathway to mediate and initiate rescue from apoptosis induced by serum deprivation, and JNK/SAPK activation along with inhibition of MAPK (ERK) are required for modulating and inducing apoptosis (31). The MAPK pathways have also been found to be necessary for CT-1 effects on promoting survival of serum-deprived cardiac myocytes and blocking MAPK activation by transfection of a dominant-negative mutant MEK or by treatment with PD098059 inhibited the survival effect of CT-1 (27). Furthermore, in the present report, we observed, even in the presence of serum, induction of apoptosis in RAW 264.7 cells upon blockade of the MAPK signaling pathways either by a dominant-negative mutant of MAPK (ERK) (Fig. 6B) or by MEK1 inhibitor, PD098059 (Fig. 7A). Thus, based on our studies and those previous studies, it is plausible that the MAPKs (ERK) represent a common pathway targeted by anti-apoptotic cytokines to promote cell survival.
It will be of great interest to determine whether similar cytokine-induced MAPK-dependent signaling pathways operate in vivo to promote cell survival, and the present findings may potentially have important clinical implications. Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent phagocytic cells and represents a critical process in tissue remodeling, regulation of immune response, or resolution of inflammatory reactions (32). The importance of TGF-␤ 1 in the regulation of inflammation is well demonstrated by the observations that TGF-␤ knock-out mice have severe and generalized inflammatory disorders (33,34). We have identified TGF-␤ 1 as an inhibitor of apoptosis in cultured macrophages and may serve as a cell survival factor via the MAPK-dependent pathway. This would provide macrophages with a cellular defense mechanism to be selectively spared from toxicity and cell death, a process that would be critical in the resolution of inflammation. Survival of macrophages is required to perform their duties of phagocytosis and elimination of adjacent harmful or injured cells, including lymphocytes, that have undergone apoptosis, and disorders that compromise macrophage survival could contribute to chronic inflammatory diseases.