Abstract
Stimulation of PPARγ1 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)γ1. 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 αβ 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 PPARγ1 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 PPARγ1. Thus, this ROCK-mediated clustering of integrin complexes is not needed for the effects of TCDD on Erk and PPARγ1. A minimal cholesterol depletion with β-methylcyclodextrin attenuated TCDD effects on PPARγ1 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 αβ integrin complexes.
Footnotes
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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, β-methylcyclodextrin; PBS, phosphate-buffered saline; MEK, mitogen-activated protein kinase kinase; Erk, extracellular signal-regulated kinase; PD98059, 2′-amino-3′-methoxyflavone; ROCK, Rho kinase.
- Received May 18, 2006.
- Accepted September 6, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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