A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis - PubMed (original) (raw)

A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis

I C Ho et al. Proc Natl Acad Sci U S A. 1998.

Abstract

NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.

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Figures

Figure 1

Figure 1

FSE2 element binds NFAT proteins. (A) The 5′ upstream promoter region of the aP2 gene with NFAT and AP-1 binding sites is highlighted and nucleotide sequence comparisons of NFAT/AP-1 binding sites in the aP2, IL-4, and IL-2 promoters are shown. (B) EMSA using T lymphocyte nuclear extracts and radiolabeled probe containing the FSE2 element (positions −127 to −101).

Figure 2

Figure 2

NFAT/AP-1 proteins are present in differentiated adipocytes and bind the FSE2. (a). Western blot analysis of cell extracts from differentiated 3T3L1 cells with antibodies to NFATp and NFAT3 is shown. (b) Western blot analysis of extracts prepared from the mesenteric fat of NFATp −/− and control NFAT +/+ mice with antibodies to NFAT3 is shown.

Figure 3

Figure 3

NFATp binds to FSE2 DNA only in differentiated adipocytes. EMSA and supershift experiments were performed with nuclear extracts from either undifferentiated (lane 5) or day 4 differentiated (the remaining lanes) 3T3-L1 cells, an FSE2 radiolabeled probe, and antibodies to NFATp and AP-1 proteins.

Figure 4

Figure 4

NFATp transactivates the aP2 promoter in adipocytes. (A) 3T3L1 cells were cotransfected with 20 μg of an aP2-CAT reporter (positions −168 to +20) and 20 μg of an NFATp expression plasmid (pRep4 NFATp) or control pRep4 plasmid in the presence or absence of phorbol 12-myristate 13-acetate (2.5 nM) and ionomycin (2 μM). CAT activity determined 48 hr later. (B) The experiment in A was repeated with either the wild-type aP2-CAT reporter or an aP2 reporter with a mutation in the NFAT site. The data were normalized to the activity of the pRep control vector (=1). One representative experiment of three is shown.

Figure 5

Figure 5

CsA inhibits adipocyte differentiation. (a) Undifferentiated 3T3-L1 cells. (b_–_d) T3L1 cells were differentiated for 6 days in the absence of control solvent (b), the presence of control solvent (c), or in CsA (10 μg/ml) (d) and stained with red oil O. The 3T3-L1 cells were differentiated in the presence of CsA (10 μg/ml) added at day 0 (d), day 1 (e), day 2 (f), day 3 (g), or day 4 (h).

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