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2,3,7,8-Tetrachlorodibenzo-p-dioxin and Epidermal Growth Factor Cooperatively Suppress Peroxisome Proliferator-Activated Receptor-1 Stimulation and Restore Focal Adhesion Complexes during Adipogenesis: Selective Contributions of Src, Rho, and Erk Distinguish These Overlapping Processes in C3H10T1/2 Cells

Department of Pharmacology, Medical Science Center, University of Wisconsin-Madison, Madison, Wisconsin

Received May 18, 2006; accepted September 6, 2006

	Abstract

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Stimulation of PPAR1 and adipogenesis in multipotential C3H10T1/2 cells by the combination of dexamethasone and 3-isobutyl-1-methylxanthine (DM) is suppressed by 2,3,7,8 tetrachlorodibenzodioxin (TCDD) (10 nM). This suppression requires sustained activation of extracellular signal-regulated kinase (Erk)1/2. We show that it arises from an effect of TCDD on epidermal growth factor (EGF) signaling. DM initiates an early loss of cell adhesion that is reversed by this TCDD/EGF synergy. Src kinase activity was completely essential for adhesion restoration, sustained Erk activation, and suppression of peroxisome proliferator-activated receptor (PPAR)1. MEK/Erk activity did not contribute, however, to TCDD-induced adhesion. Stimulation of adhesion may therefore precede elevation of Erk. Adhesion is produced by interaction of integrins with extracellular matrix proteins and subsequent Src-mediated phosphorylation of focal adhesion kinase (FAK, Tyr576/577) and paxillin (Tyr118). TCDD enhanced the steady state Src-mediated phosphorylation of FAK but not of paxillin. Protein tyrosine phosphatase (PTPase) inhibition by orthovanadate (OVA) showed that this Src activity is highly restricted by PTPases. Partial inhibition of PTPases by OVA mimicked TCDD in producing EGF- and Src-dependent effects on cell adhesion and PPAR1 suppression. TCDD may therefore induce a protein that enhances Src effectiveness at adhesion sites. Rho kinase (ROCK) inhibition blocked TCDD/EGF stimulation of clustered focal adhesion complexes without affecting either sustained Erk activation or suppression of PPAR1. Thus, this ROCK-mediated clustering of integrin complexes is not needed for the effects of TCDD on Erk and PPAR1. A minimal cholesterol depletion with -methylcyclodextrin attenuated TCDD effects on PPAR1 and Erk activation. TCDD intervention is therefore linked to extracellular proteins. It indicates that TCDD-enhanced stimulation of EGF signaling to Erk may derive from the initial integrin complexes.

The adipocyte differentiation process is inhibited by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent activator of the aryl hydrocarbon receptor (AhR). AhR is a member of the helix-loop-helix/periodicity-aryl hydrocarbon receptor nuclear translocator-simple-minded (PER/ARNT/SIM) nuclear receptor family that heterodimerizes with a related protein ARNT when activated by TCDD (Safe, 2001). This activation induces a select few genes in 10T1/2 cells including CYP1A1 and CYP1B1 (Hanlon et al., 2005b). In mice treated with higher doses of TCDD than that for inducing CYP1A1, an overall loss of body fat and weight (wasting syndrome) accompanied by increased circulating triglycerides is seen (Liu and Matsumura, 1995). Previous work has characterized a TCDD-mediated inhibition of adipocyte differentiation in 3T3-L1 preadipocytes (Phillips et al., 1995). TCDD prevents the initial stimulation of PPAR and later regulators but does not affect the expression of early response genes, including C/EBP (Phillips et al., 1995; Chen et al., 1997).

10T1/2 cells differentiate rapidly without cell division and without the mitotic stimuli provided by serum renewal or insulin (Cho and Jefcoate, 2004). We have used this characteristic of the 10T1/2 cells to completely separate gene expression associated with mitotic regulation of 10T1/2 cells from the regulation of differentiation (Hanlon et al., 2005a). In this study, we have focused on simple stimulation of the combination of dexamethasone and 3-isobutyl-1-methylxanthine (DM) without serum renewal (DM/URS).

Our previous studies have shown that TCDD synergizes with growth factors, including EGF and fibroblast growth factor in the suppression of PPAR1 expression (Hanlon et al., 2003). This synergy was proportional to the activation of Erk and completely dependent on this activity. We tested these adipogenic responses for their sensitivity to TCDD and EGF. Using microarray analysis, 30 to 40 of the most robust adipogenic responses are, like PPAR, blocked cooperatively by the combination of TCDD and EGF treatments (Hanlon et al., 2005a) (Table 1).

EGF activation of Erk through the MAP kinase pathway is typically transient because of the activation of the opposing MAP kinase phosphatase (Camps et al., 2000). We have found that TCDD enhances cell adhesion (Hanlon et al., 2005a). Cell adhesion has been linked to the prolonged activation of MAP kinase that seems necessary for TCDD suppression of PPAR1. Cell adhesion has been strongly implicated in the regulation of adipogenesis. In addition to the cell rounding in the first 24 h of adipogenesis (Spiegelman and Farmer, 1982; Hausman et al., 1996), enhanced adhesion of 3T3-L1 cells on fibronectin diminishes differentiation (Castro-Munozledo et al., 1987; Kamiya et al., 2002). The actin-binding protein enc1 is involved in both morphology changes and differentiation during adipogenesis (Zhao et al., 2000). In fat tissue, extensive deposition of ECM is also evident (Nakajima et al., 1998).

Previous work has shown that TCDD action on adipogenesis depends on Src kinase. It is noteworthy that responses are diminished in Src-/- mice and mouse embryonic fibro-blasts derived from them (Vogel and Matsumura, 2003). Integrin heterodimers initiate cell adhesion signaling through interaction with extracellular matrix proteins which recruit focal adhesion kinase (FAK) and adaptor proteins such as paxillin and CAS, which provide connections initiated by vinculin to F-actin stress fibers (Turner, 2000a). Src is a crucial mediator for these integrin complexes and intracellular signaling to the nucleus. More extended focal adhesion complexes associated with cell spreading derive from extensive clustering of integrin complexes with FAK, paxillin, and vinculin. This clustering is produced by Rho kinase-mediated cross-linking of F-actin stress fibers with myosin 2 (Defilippi et al., 1999).

View larger version (20K): [in this window] [in a new window]   Fig. 1. TCDD and EGF cooperatively suppress the DM-induced PPAR expression in a Src and Erk dependent manner. A, experimental scheme. B, time course of DM-induced PPAR expression. C3H10T1/2 cells were either untreated (UT) or treated with Dex and IBMX (DM) as described under Materials and Methods for 12, 24, and 48 h. PPAR1 and -2 expression was analyzed by Western blotting using PPAR antibody; results shown are representative of at least two experiments. C, TCDD and EGF cooperatively suppress DM-induced PPAR1 expression. Cells were treated with TCDD (10 nM) or EGF (10 nM) alone, and TCDD+EGF in combination (TCDD/EGF) in the presence of DM for 24 h. D, the suppression of PPAR1 by TCDD/EGF is reversed by the inhibition of Src and Erk. Src inhibitor PP2 (1 µM) and MEK inhibitor U0126 (10 µM) were added with TCDD/EGF, and the expression of PPAR1 was analyzed 24 h later. In C and D, the relative level of PPAR1 versus that of DM induction was expressed as mean ± S.E.M. from triplicate samples. *, p < 0.05, Student's t test.

	Materials and Methods

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Materials. MEK inhibitor U0126 was from Promega (Madison, WI). Src inhibitor PP2 and Rho kinase inhibitor Y27632 were from Calbiochem (San Diego, CA). IBMX, dexamethasone, sodium orthovanadate, -methylcyclodextrin (MCD), and EGF were from Sigma Chemical (St. Louis, MO). Dulbecco's modified Eagle's medium/Ham's F-12 medium was from Invitrogen (Auckland, NZ). Fetal bovine serum was from Atlanta Biologicals (Norcross, GA). Trypsin (0.25%) and PBS were from Mediatech (Herndon, VA). The PPAR antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The mouse monoclonal paxillin antibody was purchased from BD Biosciences PharMingen (San Jose, CA). The anti-phospho (Tyr118) paxillin antibody, anti-phospho (Tyr416) Src antibody, total Src antibody, anti-phospho (Tyr576/577) FAK antibody, total FAK antibody, and anti-phospho (Thr202/Tyr204) Erk were purchased from Cell Signaling Technology (Danvers, MA). The Alexa Fluor 488 phallotoxin, the Rhodamine Red-X goat anti-mouse IgG, and the ProLong Antifade Kit were purchased from Invitrogen.

Cell Culture. Mouse pluripotent C3H10T1/2 cells with passage number less than 15 were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% fetal bovine serum and penicillin-streptomycin (10 IU/ml and 10 µg/ml, respectively). Cells were cultured in six-well plates with initial plating density of 3 x 10⁵ cells/well. For differentiation, cells were induced with 1 µM Dex and 0.5 mM IBMX under the unrenewed serum condition (URS). TCDD (10 nM) was added to cells 48 h before induction and replaced with fresh TCDD at time 0 when Dex and IBMX were added. In the experiments regarding TCDD and EGF cooperation, EGF (10 nM) was added at time 0. For the experiments testing the role of Src inhibitor PP2 (1 µM) and MEK inhibitor U0126 (10 µM), these two inhibitors were also added to cells at time 0 during differentiation.

Western Blotting. Cells were treated as desired, and then the media was removed and cells were washed twice with PBS. Total proteins were extracted using radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 1% Nonidet P-40, 0.1% SDS, 50 mM Tris base, pH 7.4, 0.25% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail from Sigma. Total cell lysates were heated at 100°C for 5 min. Total protein (60-70 µg), determined by BCA kit from Pierce (Rockford, IL) was loaded on 10% SDS-polyacrylamide gels and separated by electrophoresis, then were transferred to a nitrocellulose membrane. After blocking, this membrane was incubated with the primary antibody of interest at 4°C overnight. The target proteins were then detected by horseradish peroxidase-conjugated secondary antibodies, and signals were visualized by adding ECL Western Blotting Detection Reagents from GE Healthcare (Little Chalfont, Buckinghamshire, UK). The intensities of bands films were quantified using software ImageQuant 5.2. The statistical analysis of significance was performed by Student's t test.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Effects of Src inhibition on acute Erk1/2 activation by EGF and sustained Erk1/2 activation by TCDD/EGF. A, experimental scheme. B, the presence of TCDD (10 nM) or DM does not alter the acute Erk activation by EGF (10 nM). C, the acute EGF-induced Erk1/2 activation is sensitive to MEK inhibition but not to Src inhibition to the same degree. Cells were pretreated with either the MEK inhibitor U0126 (1 and 10 µM) or the Src inhibitor PP2 (0.1, 1, and 10 µM) for 20 min, then EGF was added for 20 min. D, TCDD/EGF causes a sustained Erk1/2 activation at 12 and 24 h after treatment in the presence of DM, which was not observed when cells were treated with either TCDD (10 nM) or EGF (10 nM) alone. E, the level of TCDD/EGF-induced sustained Erk1/2 activation is low compared with the acute Erk1/2 activation at 20 min. F, the sustained Erk1/2 activation induced by TCDD/EGF at 24 h is suppressed by PP2 (1 µM) and U0126 (10 µM). In the above experiments, cells were treated as indicated, and the total proteins were harvested at the indicated time points, and phosphorylation of Erk1/2 (Thr202/Tyr204) is analyzed by phosphospecific antibody using Western blotting described under Materials and Methods. The results shown above are representative of at least two experiments.

Cell Treatment with MCD. MCD with different concentrations (0.1, 1, or 4 mM) was added to cells 5 h before differentiation induction. At time 0, C3H10T1/2 cells were induced to differentiation as described above with replacement of fresh MCD in the medium. Cells were treated for 24 h, and the total proteins were harvested to study the effect of MCD on PPAR1 expression and Erk phosphorylation.

Src Kinase Assay. After cells were treated with TCDD and EGF under URS/DM conditions, the kinase activity of Src was assayed using a Universal Tyrosine Kinase Assay kit from Takara Biotech, Inc. (Shiga, Japan). In brief, the Src kinase was first immunoprecipitated by the Src antibody and diluted. Then the diluted samples were added to a 96-well plate which was precoated with synthesized peptide substrate (poly Glu-Tyr). The reaction was started by adding ATP-2Na, and the phospho-tyrosine was detected by an anti-phosphotyrosine (pY20)-HRP antibody provided by the kit. Then, the substrate of HRP was added to each reaction, and the kinase activity was measured by the absorbance at 450 nM with the µQuant plate reader from Bio-Tek Instruments (Winooski, VT).

Statistical Analysis. In the experiments of triplicate samples, the result was shown by mean ± S.E.M. Statistical significance between different treatments was analyzed by Student's t test.

	Results

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TCDD and EGF Cooperatively Inhibit the Expression of PPAR1 When Stimulated by Dex/Mix under URS Condition; MEK and Src Are Key Mediators. In previous studies, we have shown that PPAR1 expression and subsequent adipogenesis are fully induced by DM without insulin and also that omission of the serum renewal (URS) with this addition does not lessen these responses (Cho and Jefcoate, 2004). These two changes completely remove any initial stimulation of the MEK/Erk pathway and mitogenesis but maintain a modest increase in DNA synthesis.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Effect of TCDD treatment on Src activation. A, experimental scheme; B, Src phosphorylation on Tyr416 is enhanced by PP2 during the acute EGF treatment. Cells were pretreated with either the MEK inhibitor U0126 or the Src inhibitor PP2 for 20 min; EGF was then added for 20 min. The phosphorylation of Src Tyr416 was analyzed using phosphor-specific antibody as described under Materials and Methods. C, PP2 alone causes an increase of Src Tyr416 phosphorylation. Cells were treated with PP2 (0.1, 1, and 10 µM) as in B, or for 24 h, without EGF. The phosphorylation of Src Tyr416 was analyzed as described in B. D, EGF increases Src kinase activity at 24 h in the presence or absence of TCDD. After the treatments indicated in the chart, Src kinase activity was analyzed as described under Materials and Methods; results shown are from duplicated samples and representative of the trend from other experiments. E, EGF increases the phosphorylation of Src Tyr416 at 24 h in the presence or absence of TCDD. Cells were treated as indicated; the phosphorylation of Src Tyr416 at 12 and 24 h was analyzed by the same method in B.

The cooperation between TCDD and EGF in this suppression was completely reversed by the MEK inhibitor U0126 (10 µM) (Fig. 1D) at levels that completely inhibit the phosphorylation of Erk (see Fig. 2). MEK inhibitors at higher concentrations can bind to AhR (Andrieux et al., 2004). Else-where, we have shown that inhibition of MEK/Erk by either U0126 or PD98059 does not change the parallel TCDD induction of CYP1B1, a marker protein for AhR activation (Hanlon et al., 2003) (X. Liu and C. Jefcoate, unpublished data). EGF also does not enhance the stimulation of an AhR-selective xenobiotic response element-luciferase reporter under these conditions (Hanlon et al., 2005b) even though MAP kinases directly enhance ARNT/AhR activity in some cell types (Tan et al., 2004). In addition, we have shown previously that inhibitor effects on Erk phosphorylation closely match the reversal of PPAR1 synthesis and of subsequent adipogenesis (Hanlon et al., 2003). Thus, the reversal of TCDD/EGF effects by U0126 comes from the interaction with MEK rather than from a displacement of TCDD from AhR.

Several reports have implicated Src kinase as a participant in TCDD activity (Vogel and Matsumura, 2003; Vogel et al., 2003; Hoelper et al., 2005). In Fig. 1D, we also show that a low level of a selective Src kinase inhibitor PP2 (1 µM) also completely prevented the inhibitory effect of the TCDD/EGF combination.

View larger version (42K): [in this window] [in a new window]   Fig. 4. DM treatment induces loss of cell adhesion. A, time course of adhesion loss in C3H10T1/2 cells, as shown by disruption of focal adhesion complex (paxillin) and actin stress fibers (phalloidin). Cells were either untreated (UT) or treated with DM for 12, 24, or 48 h, then immunostained using anti-paxillin antibody for paxillin focal adhesion, phallotoxin for actin stress fiber and DAPI for nuclei as described under Materials and Methods; pictures shown for each treatment reflect cells from the same field. B and C, time courses of tyrosine dephosphorylation of paxillin and FAK under DM treatment. Cells were treated as described in A, and phosphorylations of paxillin (Tyr118) and FAK (Tyr576/577) were analyzed by phosphospecific antibodies as described under Materials and Methods.

We have shown previously that delayed inhibition of Erk activation even after the initial 6 h is fully effective in preventing TCDD suppression of PPAR1 and adipogenesis (Hanlon et al., 2003). This suggests that TCDD activity depends on prolonged Erk activity rather than the high transient Erk stimulation that is reversed after 20 min by stimulation of the MAP kinase-activated phosphatase (Camps et al., 2000). We therefore focused on sustained Erk phosphorylation, which maintains a steady state between 6 and 24 h (Fig. 2E). In contrast to the early EGF-stimulated peak, TCDD substantially stimulated this p-Erk steady-state level up to approximately 10% of the 20-min peak (Fig. 2D). In the presence of DM, EGF could sustain p-Erk at only approximately the basal level, which was otherwise completely suppressed by DM. In contrast to the acute stimulation, 1 µM PP2 substantially blocked this stimulation of sustained Erk phosphorylation by TCDD/EGF (Fig. 2F). The time period for this Erk activity is consistent with the delayed period (6-12 h) required for Erk mediation of the PPAR1 suppression (Hanlon et al., 2003; Cimafranca et al., 2004). The high sensitivity to PP2 of the sustained pErk also matches the sensitivity of the TCDD/EGF suppression of PPAR1 (Fig. 1).

Src Is Acutely Regulated through Tyr416 Phosphorylation That Is Determined through a Src-Dependent Feedback Loop. Src kinase activity is greatly enhanced by phosphorylation adjacent to the catalytic site at Tyr416. EGF did not increase the phosphorylation of Src during the period of maximum Erk phosphorylation (initial 20 min) (Fig. 3B). Src phosphorylation, however, is tightly coupled to PTPase activity (Roskoski, 2005). EGF slowly increased this steady-state Tyr416 phosphorylation between 12 and 24 h, but this was not affected by TCDD (Fig. 3E). The kinase activity of immunoprecipitated Src on a synthetic peptide (poly-Glu-Tyr) paralleled the Tyr416 phosphorylation (Fig. 3D). The phosphorylation of the inhibitory Src regulatory site (Tyr527) was relatively insensitive to EGF, TCDD, and PP2 (data not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 5. TCDD and EGF cooperate to restore the DM-induced loss of adhesion. A, Effects of TCDD and EGF on focal adhesion complexes (paxillin) and actin stress fibers (phalloidin). Cells were either untreated (UT) or treated with TCDD (10 nM), EGF (10 nM), or TCDD+EGF in the presence of DM for 24 h, and then were immunostained as described under Materials and Methods. Pictures shown for each treatment reflect the same field of cells. B and C, effects of TCDD and EGF on the phosphorylation of paxillin (Tyr118) and FAK (Tyr576/577). Cells were treated as described in A; total proteins were harvested at 24 h, and the phosphorylations of paxillin (Tyr118) and FAK (Tyr576/577) were analyzed using phospho-specific antibodies as described under Materials and Methods. The levels of phosphorylation of paxillin and FAK versus control were expressed as mean ± S.E.M from triplicated samples. *, p < 0.05, Student's t test.

DM Induces Loss of Focal Adhesion Complexes and F Actin Stress Fibers. To determine how Src is participating in TCDD suppression, we examined the established participation of Src in cell adhesion mediated by the interaction of integrins with extracellular matrix proteins (Parsons et al., 2000). Cell rounding caused by a loss of surface and intercellular adhesion is seen in mouse embryo fibroblasts at an early stage in differentiation (Spiegelman and Farmer, 1982; Hausman et al., 1996). We first characterized the time course of the DM-induced morphological changes in relation to the increases in PPAR1 described in Fig. 1. Immunostaining of paxillin foci identifies the number and location of integrin associated focal complexes. Fluorescein isothiocyanate-labeled phalloidin binding to F-actin visualizes the length and distribution of stress fibers that cross-link integrin-associated focal complexes. Figure 4A shows that DM decreased the number of paxillin foci that are visualized as white or red dots. These foci decreased appreciably after 12 h, although few were detectable after 24 and 48 h. In the untreated cells, F-actin stress fibers distribute from nucleus to plasma membrane and form extended structures under the plasma membrane that produce larger clustered focal adhesion complexes. Long projections extending between cells are also visible. These intracellular stress fibers decline appreciably by 12 h, although in some cells more than others (Fig. 4A). Because paxillin foci and F-actin stress fibers are visualized in the same cells, it is apparent that retention of F-actin fibers is associated with residual paxillin foci.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Effects of Src inhibition (PP2) and MEK inhibition (U0126) on TCDD/EGF-induced restoration of cell adhesion. A, PP2, but not U0126, blocks the TCDD/EGF-induced restoration of adhesion complexes (paxillin) and stress fibers (phalloidin). Cells were treated as indicated in the figure and immuno-stained for paxillin and phalloidin as described under Materials and Methods; pictures shown for each treatment reflect cells from the same field. B, restoration of paxillin Tyr118 phosphorylation is sensitive to PP2, not U0126, whereas both inhibitors suppress FAK Tyr576/577 phosphorylation. After the indicated treatments for 24 h, total proteins were harvested, and the Tyr118 phosphorylation of paxillin and the Tyr576/577 phosphorylation of FAK were analyzed using the phosphospecific antibody as described under Materials and Methods. Results are representative of at least two experiments.

TCDD and EGF Cooperatively Prevent the DM-Induced Loss of Focal Adhesion Complexes and F Actin Stress Fibers. Figure 5A shows that TCDD and EGF alone were largely ineffective in preventing these losses of paxillin foci and actin stress fibers after 24 h of DM treatment. By contrast, the TCDD/EGF combination restored most of the morphology characteristics seen in the untreated cells (Fig. 5A). Additional changes have also occurred because the F-actin fibers project less between TCDD/EGF-treated cells than in the untreated cells. Major differences between untreated cells and TCDD/EGF-treated cells are indicated by our previous expression profiling of the cells under these two conditions (Hanlon et al., 2005a). The adipogenic stimulus produced substantial losses in the expression of mRNA corresponding to extracellular matrix and structural proteins, including F actin, that were either unaffected or enhanced by TCDD/EGF treatment (Hanlon et al., 2005a).

The DM-induced loss of phosphorylation of paxillin (Tyr118) was appreciably restored by EGF at 24 h (Fig. 5B). However, because EGF did not restore the focal complexes, this increased paxillin phosphorylation is evidently insufficient for the increase in adhesion. The combination of TCDD and EGF, which restored the adhesion complexes and stress fibers, did not further increase paxillin phosphorylation. This paxillin phosphorylation is therefore insufficient to restore adhesion without TCDD induction of some additional participant. EGF did not increase FAK Tyr576/577 phosphorylation (Fig. 5C), which is therefore unlikely to mediate the EGF stimulation of paxillin phosphorylation. The combination of TCDD and EGF significantly increased the FAK phosphorylation compared with the single treatments (p < 0.05, n = 3) (Fig. 5C). Although this parallels the restoration of adhesion, as noted in Fig. 4, the slow DM-induced loss of this FAK phosphorylation appeared after the adhesion change.

Src Kinase, but Not Erk, Is Required for the TCDD and EGF Cooperation in Restoring the DM-Induced Loss of Cell Adhesion changes. Src kinases participate in the signaling between integrin complexes by mediating tyrosine phosphorylation of intracellular partners including FAK and growth factor receptors (Schlaepfer et al., 1994). When appropriately activated by ECM binding, intracellular regions of 1 integrins interact with FAK, leading to auto-phosphorylation of Tyr397. This site then interacts with the SH2 domain of Src to initiate further phosphorylations, including Y-576/577 and paxillin Tyr118. We have used paxillin pTyr118 and FAK pTyr576/577 as indicators of Src kinase activity in the focal adhesion complexes.

Figure 6A shows that restoration of paxillin foci and F-actin stress fibers by the TCDD/EGF combination was blocked by 1 µM PP2 and is therefore dependent on Src kinase activity. The TCDD/EGF restoration of paxillin and FAK phosphorylations was also completely blocked by PP2 (1 µM) (Fig. 6B). This potent inhibition by PP2 corresponded closely to the inhibition of sustained Erk phosphorylation and the reversal of PPAR1 suppression (Figs. 1D and 2F). The synergistic effects of TCDD and EGF that restore adhesion, sustain the Erk activation, and suppress PPAR1 expression are therefore likely to be mechanistically linked.

These effects of the TCDD/EGF combination on cell morphology were insensitive to U0126 (Fig. 6A), thus providing further evidence that MEK/Erk does not participate in the initial TCDD induction process that controls adhesion. However, FAK phosphorylation was blocked by U0126 (Fig. 6B). As noted above, this effect of TCDD/EGF on FAK Tyr576/577 phosphorylation seems to be a later step that results from the increased adhesion. The effect of U0126 demonstrates that FAK phosphorylation needs the MEK/Erk activity, which increases after the adhesion restoration. Recruitment of Erk to focal adhesion complexes has been reported (Fincham et al., 2000).

View larger version (40K): [in this window] [in a new window]   Fig. 7. Inhibition of Rho kinase blocks the restoration of cell adhesion by TCDD/EGF but does not affect either the suppression of PPAR1 expression or the sustained Erk1/2 activation. A, the suppression of DM-induced PPAR1 expression and the sustained Erk1/2 activation by TCDD/EGF are not affected by Rho kinase inhibitor Y27632 (10 µM). Cells were treated as indicated, total proteins were harvested at 24 h, and PPAR1 expression was analyzed by Western blotting. The level of PPAR1 versus DM induction is expressed as mean ± S.E.M from triplicate samples. *, p < 0.05, Student's t test. The phosphorylation of Erk1/2 (Thr202/Tyr204) was analyzed by phosphospecific antibody using Western blotting as described under Materials and Methods. B, inhibition of Rho kinase by Y27632 blocks TCDD/EGF effects on cell adhesion during DM treatment. Cells were treated with TCDD/EGF+DM in the presence or absence of Y27632 (10 µM) for 24 h. Paxillin focal adhesion complexes and phalloidin stress fiber were fluorescently stained as described under Materials and Methods. Pictures shown are from the same field of cells.

Figure 7A shows that the selective ROCK inhibitor Y27632 (10 µM) blocked neither the TCDD/EGF suppression of PPAR1 nor the sustained Erk activation but nevertheless inhibited the restoration of cell adhesion by TCDD/EGF. This is visualized by the effect of TCDD/EGF on the distribution of paxillin and F actin stress fibers (Fig. 7B). This result indicates that TCDD/EGF maintenance of extended focal contacts and stress fibers are not needed for either the PPAR1 suppression or the sustained Erk activation produced by TCDD/EGF. Therefore, ROCK and the extended clustering of focal integrin complexes are not involved in either of these processes. This parallel further supports the causal relationship between sustained Erk activation and the suppression of PPAR1. By contrast, Y27632 (10 µM) prevented the increases in the phosphorylations of FAK (Tyr576/577) and paxillin (Tyr118) produced by TCDD/EGF (X. Liu and C. Jefcoate, unpublished data). These effects of Y27632 confirm that these steady-state phosphorylation levels are set by the extended adhesion structures.

Orthovanadate (OVA), a General PTPase Inhibitor, Restores EGF Stimulation of FAK (Tyr576/577) Phosphorylation and Enhances Paxillin (Tyr118) Phosphorylation. The steady-state levels of paxillin (Tyr118) and FAK (Tyr576/577) phosphorylation depend on the net activities of Src and opposing PTPases. Several PTPases have been implicated in the dephosphorylation of FAK and paxillin, including SHP2, PEST-PTPase, and PTP1B (Oh et al., 1999; Jin et al., 2000; Turner, 2000b). These steady-state measurements, however, may not provide a good indicator of onward kinase signaling because the PTPases also compete with the recruitment of SH2 domains. This includes proteins such as Grb2, which activates the MEK/Erk pathway (Fig. 8A, scheme). PTPase activity may actually target excess pTyr that exceed the availability of SH2 proteins. We therefore varied the concentrations of a general PTPase inhibitor OVA to test the impact of PTPase activity on Src-dependent phosphorylations of FAK and paxillin. We also examined effects on both adhesion characteristics and PPAR1 expression. Figure 8B shows that 24 h after DM, several steady-state phosphorylations were modestly increased by 1 µM OVA and substantially increased by 5 µM OVA.

View larger version (51K): [in this window] [in a new window]   Fig. 8. Inhibition of PTPase by low levels of OVA cooperates with EGF to restore FAK and paxillin phosphorylation and to suppress PPAR1 expression after DM treatment. A, proposed model for the role of PTPase played in the activation of FAK and paxillin by Src kinases. The Src generation of the activated pTyr forms of FAK and paxillin is limited by more active PTPases. This results in a low steady-state phosphorylation; the activity of SH2 effectors such as Grb2 depends on competition with the PTPases for pTyr sites. B, low levels of OVA produce modest effects on the overall phosphorylation status of cells in the presence of DM. Cells were treated with increasing concentrations of OVA (0.25, 0.5, 1, 5 µM) in the presence of DM for 24 h. The overall phosphorylations were analyzed by anti-pTyr100 antibody as described under Materials and Methods. C, low level of OVA (1 µM) cooperates with EGF (10 nM) to restore the phosphorylation of paxillin and FAK in the presence of DM. Cells were treated as indicated for 24 h; the phosphorylation of paxillin (Tyr118), FAK (Tyr576/577) was analyzed by Western blotting as described under Materials and Methods.D, low levels of OVA (1 µM) cooperates with EGF (10 nM) to suppress the DM-induced PPAR1 expression, which is not seen in the cells treated with OVA (1 µM) and TCDD. Cells were treated as indicated for 24 h. PPAR1 expression was analyzed by Western blotting as described under Materials and Methods. E, the suppression of PPAR1 by OVA/EGF is sensitive to the inhibition of Src and Erk. Src inhibitor PP2 (1 µM) or Erk inhibitor U0126 (10 µM) was added with OVA/EGF for 24 h, and the suppressed PPAR1 expression was restored by both inhibitors. In D and E, the level of PPAR1 versus that of DM induction was expressed as mean ± S.E.M. from triplicate samples. *, p < 0.05, Student's t test.

The modest inhibition of PTPases by OVA (1 µM) in combination with EGF suppressed PPAR1 in much the same way as the combination of TCDD with EGF (Fig. 8D). This suppression of PPAR1 depended on both Src and Erk, as evidenced by reversal effects of, respectively, PP2 (1 µM) and U0126 (10 µM) (Fig. 8E). OVA (1 µM) had little effect alone and only slightly increased the effectiveness of TCDD. It is noteworthy that EGF and 1 µM OVA not only restore adhesion but also sustain Erk phosphorylation after DM treatment (X. Liu and C. Jefcoate, manuscript in preparation). Thus, this partial inhibition of PTPase activity by OVA synergizes with EGF in essentially the same way as processes induced by TCDD.

View larger version (29K): [in this window] [in a new window]   Fig. 9. Mild MCD treatment partially reverses the TCDD/EGF-induced suppression of PPAR1 and the sustained Erk activation. A, experimental scheme. B, low level of MCD (0.1 mM) partially restores the TCDD/EGF-induced suppression of PPAR1 with partial blocking of the sustained Erk1/2 activation. Cells were treated as indicated in the experimental scheme, in the presence or absence of TCDD/EGF (TE). Total proteins were harvested at 24 h; PPAR1 expression and phosphorylation of Erk1/2 (Thr202/Tyr204) were analyzed by Western blotting as described under Materials and Methods. The relative levels of PPAR1 were quantified by densitometry and expressed as mean ± S.E.M from triplicate experiments. *, p < 0.05, Student's t test. C, higher doses of MCD further block the TCDD/EGF-sustained Erk activation but gradually diminish the restoration of PPAR1 expression. Cells were treated as described in B, with 1 and 4 mM MCD. After 24 h, total proteins were harvested to analyze PPAR1 expression and phosphorylation of Erk1/2 (Thr202/Tyr204).

TCDD/EGF Suppression of PPAR1 Is Sensitive to -Methylcyclodextrin Treatment.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
During adipogenesis in C3H10T1/2 cells, PPAR1 increases before the appearances of C/EBP and PPAR2 (Cho et al., 2005). These nuclear factors collectively mediate insulin-dependent lipogenesis (Rosen and Spiegelman, 2000). We have shown previously that TCDD suppression of PPAR1 and of these later processes each depends on synergy with EGF activation of the MEK/Erk pathway (Hanlon et al., 2003, 2005a). A cluster of approximately 40 adipogenic gene responses each exhibit cooperative inhibition by TCDD and EGF, many of which have clear links to various aspects of cell metabolism (Table 1). This synergy functions in a relatively narrow time window immediately before the increased transcription of PPAR1. Some of these transitional gene changes, typified by those shown in Table 1, may contribute to the suppression PPAR1 (Fig. 10A). Here we show that during this time window, TCDD and EGF cooperatively enhance adhesion-associated signaling, which is linked to a sustained large increase in Erk phosphorylation. We have previously found that the level of Erk phosphorylation in early adipogenesis, when TCDD is present, is proportional to the later suppression of PPAR1 and lipogenesis (Hanlon et al., 2003). This signaling is likely to also target the expression changes produced by cooperation between TCDD and EGF shown in Table 1, some of which may target the PPAR1 promoter.

View larger version (28K): [in this window] [in a new window]   Fig. 10. Model for cooperation between TCDD and EGF in adhesion, sustained Erk activation, and suppression of PPAR1. A, after DM stimulation, a "transition" period occurring after 10 to 24 h precedes the stimulation of PPAR expression. In this period, DM induces a loss of cell adhesion and expression of transition genes (Table 1). TCDD and EGF cooperatively suppress the events occurred in this transition period, leading to suppression of DM-induced PPAR1 expression. TCDD is likely to induce a mediator, which enhances the effectiveness of Src kinase stimulation by EGF. This cooperation between the unknown mediator and Src kinase leads to the restoration of focal adhesion and the sustained Erk1/2 activation. This sustained Erk activation initiates synthesis of a suppressor of PPAR expression, which may be a member of the group of transition genes. Erk activation does not contribute directly during PPAR1 synthesis. B, possible cooperation between integrin signaling and EGF/EGFR signaling to sustain Erk activation. Interaction of ECM components leads to the aggregation of and integrin, which activates FAK, leading to Src-mediated tyrosine phosphorylation of FAK. A further Src phosphorylation step leads to sustained Erk activation through SH2 recruitment of Grb/SOS to FAK pY 576/925. EGF/EGFR enhances FAK phosphorylation through Src/CAS cross-talk.

Cell adhesion is restored synergistically by the combination of TCDD and EGF, which also maximally suppresses PPAR1 (Figs. 1 and 5). This restoration of adhesion by TCDD requires Src kinase activity, again exactly paralleling a requirement for Src activity in the suppression of PPAR1 (Figs. 1 and 6). Phosphorylation of Erk is sustained at 10% of the peak transient level of acute stimulation for more than 12 to 24 h, but only with TCDD treatment. This increase in phosphorylation of Erk is maintained throughout the time window associated with suppression of PPAR1 transcription. This window largely precedes the increase in PPAR1, which is not directly sensitive to MEK inhibition. We previously hypothesized that this period corresponds to TCDD/EGF cooperation in the synthesis of a suppressor of PPAR1 transcription (Hanlon et al., 2003). We can now add that an important contributor to this suppression process is the sustained elevation of Erk phosphorylation. This shared dependence on Src activity of enhanced cell adhesion, sustained Erk activation, and PPAR1 suppression suggests that the three processes are mechanistically linked.

In contrast to PPAR1 suppression, the MEK/Erk activity has no role in the TCDD restoration of adhesion (Fig. 6). This suggests that the TCDD-induced step initiates the adhesion response before the requirement for Erk. This separation of TCDD and Erk steps also provides the strongest evidence that Erk does not participate in PPAR1 suppression by directly modifying AhR or ARNT. However, this activation of the heterodimer occurs in other processes (Tan et al., 2004), and we find that EGF stimulation of TCDD responses during adipogenesis depends on the particular target gene. Most responses, including an xenobiotic response element reporter, are little affected by further addition of EGF, but some genes exhibit appreciable synergy (Table 1) (Hanlon et al., 2005b). The preponderance of our data points to separate contributions by TCDD induction and EGF/Src signaling to adhesion, which initiates the signaling that sustains Erk activation (Fig. 10A, scheme).

In this model, we propose that TCDD induces a protein that enhances EGF activation of Src activity within adhesion sites. Enhanced effects of EGF on the Src-mediated restoration of cell adhesion and the suppression of PPAR1 are similarly seen when TCDD is replaced by low levels of the PTPase inhibitor OVA (1 µM) (Fig. 8). This treatment inhibits PTPase activities nonspecifically by approximately 50% (data not shown) and increases the phosphorylation of Src substrates such as FAK and paxillin in adhesion sites. OVA directly enhances Src effectiveness by diminishing the reverse dephosphorylation, notably at adhesion proteins like paxillin (Fig. 8).

Despite these close parallels between the TCDD/EGF synergy in cell morphology and the effects on PPAR1 suppression and Erk activation, our data show that they are not causally related. The cross-linking of F actin fibers with myosin light chains that is controlled by ROCK directs extensive clustering of integrin adhesion complexes, which are attached to F actin via paxillin (Amano et al., 1997). Inhibition of Rho kinase (ROCK) by Y27632 prevents TCDD effects of cell morphology but retains the activation of Erk and suppression of PPAR1 expression (Fig. 7).

The high sensitivity of TCDD effects on PPAR1 and Erk to cholesterol perturbation by -MCD suggests an important role for cholesterol rafts. The ECM/integrin adhesion processes are initiated within noncaveolin cholesterol rafts and then shift to cytoplasmic sites as the extensive clustering occurs (Leitinger and Hogg, 2002; Upla et al., 2004; Williams and Lisanti, 2004). Caveolin-/- mice exhibit an intracellular accumulation of the markers of noncaveolin rafts along with a loss of adipocytes in the fat pads (Razani et al., 2002). TCDD may enhance the distribution of Src to these rafts. It is notable that glypican 1, which is highly induced by TCDD in these cells (Table 1) and locates to these lipid rafts, also activates Src (Bianco et al., 2003; Chu et al., 2004). The concentration of -MCD that reverses TCDD effects on PPAR1 and Erk (0.1 mM) does not significantly deplete total cholesterol from plasma membrane but is likely to affect cholesterol distribution. Higher concentrations of MCD (4 mM) deplete cholesterol inhibited PPAR1 expression, as previously reported in 3T3-L1 cells (Huo et al., 2003).

Adhesion in preadipocytes primarily involves fibronectin interaction with 51 integrins (Fukai et al., 1993; Kubo et al., 2000). The importance of adhesion has been emphasized by the finding that 5 integrin is expressed early and then expression switches to the 6 integrin in mature adipocytes, where laminin provides the preferred ECM (Liu et al., 2005a). An initial FAK phosphorylation (pTyr397) then recruits Src, which then further phosphorylates FAK and adaptor proteins such as paxillin, which binds F actin stress fibers (Schlaepfer et al., 1994; Giancotti and Ruoslahti, 1999; Turner, 2000a) (Fig. 10B, scheme). This activation of integrins leads to Src phosphorylation of FAK (Tyr576, Tyr925) and paxillin (Tyr118), consistent with our findings. FAK activated by Src also mediates cross-talk with the EGF receptor via CAS phosphorylation (Moro et al., 2002). This cross-talk is clearly a candidate for Src mediation of Erk activation.

Src, FAK, and paxillin play key roles in adhesion, but the correlation between their phosphorylations and cell adhesion is poor in these TCDD-mediated responses. Although FAK and paxillin phosphorylations decline substantially after adipogenic stimulation (Fig. 4), paxillin phosphorylation is restored by EGF alone without full adhesion (Figs. 5), and FAK phosphorylation is sensitive to MEK inhibition by U0126, which does not affect adhesion restoration (Figs. 5, 6). Likewise, the increase in Src Tyr416 phosphorylation and kinase activity appear only after adhesion increases. However, the effects of OVA inhibition on steady-state phosphorylation establish the far greater activity of the several PTPases, which function in adhesion sites (SHP2, PTP, PTP-1B, etc.) (Oh et al., 1999; Jin et al., 2000; Turner, 2000b). Nevertheless, increased Src activity can increase downstream processes without an increased steady-state activation, if the recruitment of SH2-dependent protein mediators such as Grb2 to FAK pY sites is faster than the competing PTPase inactivation (Fig. 8). The effectiveness of these PTPases is enhanced by a negative control of Src activity during adipogenesis (Fig. 3). Inhibition of kinase activity with PP2 alone substantially increases phosphorylation of the activation site (Tyr416) adjacent to the catalytic site, largely independent of the addition of EGF. This establishes a feedback loop in which pTyr416 and Src activity are attenuated by certain PTPases that are regulated by Src kinase itself.

Although we have seen changes in Src, PP2 also inhibits other Src family kinases that participate in adipogenesis. For example, interactions between the Src-family kinase fyn located in lipid rafts with flotillin and F-actin play an important role in insulin action in mature adipocytes (Liu et al., 2005b). At this stage, we cannot exclude contributions from other Src-family members to PP2-sensitive processes that mediate the TCDD/EGF synergy.

This work establishes a persistent link between TCDD suppression of PPAR1 and the sustained Src-mediated activation of Erk kinase. This is seen under a wide range of conditions, including treatment with the selective inhibitors PP2 (for Src), U0126 (for MEK/Erk), and Y27632 (for ROCK) and cholesterol re-distribution (MCD). However, this work also establishes that the observed TCDD-induced and Src-mediated actin polymerization and extended paxillin clustering is not required for PPAR1 suppression. These morphology changes therefore occur subsequently or in parallel to the suppression pathway. Our working hypothesis is that PPAR1 suppression is mediated by a TCDD-induced early mediator protein that enhances the effectiveness of EGF to Src within the initial integrin/ECM complexes that form in cholesterol rafts. How Erk produces suppression remains a key question. We have excluded a direct phosphorylation of a PPAR1 transcriptional activator because MEK/Erk inhibition is ineffective during PPAR1 elevation (Hanlon et al., 2003). C/EBP activation that participates in PPAR transcription was not affected by TCDD (Phillips et al., 1995; Chen et al., 1997). Thus, increased expression of a suppressor, including proteins listed in Table 1, is likely to mediate the suppression of PPAR1 by TCDD. Future experiments will address the links between the TCDD and EGF stimulated changes described here including raft formation and the molecular control of PPAR1 transcription.

	Footnotes

 
ABBREVIATIONS: Dex, dexamethasone; IBMX, 3-isobutyl-1-methylxanthine; IDM, insulin/dexamethasone/3-isobutyl-1-methylxanthine; C/EBP, CCAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; DM, dexamethasone/3-isobutyl-1-methylxanthine; URS, unrenewed serum condition; MAP, mitogen-activated protein; OVA, sodium orthovanadate; ECM, extracellular matrix; FAK, focal adhesion kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; Y27632, N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride; MCD, -methylcyclodextrin; PBS, phosphate-buffered saline; MEK, mitogen-activated protein kinase kinase; Erk, extracellular signal-regulated kinase; PD98059, 2'-amino-3'-methoxyflavone; ROCK, Rho kinase.

Address correspondence to: Dr. Colin R. Jefcoate, Department of Pharmacology, University of Wisconsin-Madison, 1300 University Ave., Madison, WI 53706. E-mail: jefcoate{at}wisc.edu

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