2,3,7,8-Tetrachlorodibenzo-p-dioxin and Epidermal Growth Factor Cooperatively Suppress Peroxisome Proliferator-Activated Receptor-γ1 Stimulation and Restore Focal Adhesion Complexes during Adipogenesis: Selective Contributions of Src, Rho, and Erk Distinguish These Overlapping Processes in C3H10T1/2 Cells

Abstract

Stimulation of PPAR␥1 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 PPAR␥1 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 PPAR␥1. Thus, this ROCKmediated clustering of integrin complexes is not needed for the effects of TCDD on Erk and PPAR␥1. A minimal cholesterol depletion with ␤-methylcyclodextrin attenuated TCDD effects on PPAR␥1 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.Extensive studies on adipocyte differentiation have used mouse embryonic fibroblastic cell models, including 3T3-L1 and 3T3-422A, which are already committed to the adipocyte lineage. C3H10T1/2 (10T1/2), a pluripotent progenitor cell type that is similar to mouse embryo fibroblasts, has also been used (Alexander et al., 1998). This cell type develops into fat, bone, or muscle cells according to the stimulus (Ntambi and Young-Cheul, 2000). In general, differentiation of 3T3-L1 and 10T1/2 cells is stimulated by a hormonal mixture (IDM) composed of insulin, dexamethasone (Dex), and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which is administered to confluent cells along with serum renewal (Cowherd et al., 1999). Over approximately 48 h in 10T1/2 cells, IDM cooperatively directs several Article, publication date, and citation information can be found at

Cell Adhesion and Spreading Affect Adipogenesis from Embryonic Stem Cells: The Role of Calreticulin

“…Furthermore, a number of studies showed that increased fibronectin expression and deposition diminishes adipocyte differentiation [22, 46, 47, 74, 75]. CaMKII promotes focal adhesion turnover by inducing dephosphorylation of FAK and paxillin [76], whereas recruitment of paxillin and FAK to focal adhesions inhibits adipogenesis [45, 77, 78]. This could account for the decreased recruitment of paxillin and FAK to focal adhesions in the calreticulin‐deficient ESCs that are highly adipogenic and show elevated CaMKII levels.…”

Cytoskeletal Disassembly and Cell Rounding Promotes Adipogenesis from ES Cells

“…Increased activity of CaMKII (as in the case of cytochalasin D treatment) has been shown to diminish cell adhesiveness [81][82][83], and we observe an association between activation of CaMKII and increased cell rounding [12,32,34,79]. CaMKII activation reduces cell adhesiveness by promoting dephosphorylation of focal adhesion kinase and paxillin [83], which reduces their incorporation into focal adhesions and may promote adipogenesis [11,84,85]. In contrast, nocodazole decreases the activity of CaMKII, which may have multiple, stimulatory effects on cell adhesion.…”

Hair Follicle Stem Cell-Specific PPARγ Deletion Causes Scarring Alopecia

“…The genetic or environmental mechanisms that initiate loss of PPARγ signaling in LPP are not fully understood. However, the skin as the outermost barrier of the body is exposed to various sources of environmental toxins such as dioxin or dioxin-like compounds that are known to inhibit the expression of PPARγ and all lipogenic genes that are transcriptionally activated by PPARγ (Liu and Jefcoate, 2006). Dioxin-like compounds exert their biologic effects via the AhR, a ligand-dependent transcription factor.…”