Dexamethasone signaling is required to establish the postmitotic state of adipocyte development

The clonal expansion phase of 3T3-L1 adipose conversion is a distinct mitotic period during which the initiation of differentiation occurs concomitant with a discrete set of mitotic divisions. During clonal expansion, a cocktail of adipogenic hormones, including the glucocorticoid dexamethasone, induced 3T3-L1 cells to progress from postconfluent adipoblasts to postmitotic adipocytes. It is reported here that expression of the growth arrest-associated gene 2 (gas2) discriminated reversible, postconfluent growth arrest from irreversible, postmitotic growth arrest. In the absence of dexamethasone, 3T3-L1 cells underwent mitoses but failed to establish postmitotic growth arrest, as evidenced by the persistence of elevated GAS2 mRNA. Moreover, the dexamethasonedeprived 3T3-L1 cells appeared to revert to postconfluent growth arrest, as judged by (a) their ability to reenter logarithmic growth under permissive conditions, and (b) their ability to undergo adipose conversion when subsequently challenged with a complete cocktail of adipogenic hormones. The growth potentiating factor-encoding gene, gas6, was shown to be an immediate-early target of dexamethasone. These findings reveal the requirement for dexamethasone in establishing the postmitotic state that accompanies differentiation. In addition, the results suggest that dexamethasone signaling influences the mitotic divisions of clonal expansion by determining the nature of the growth arrest state assumed upon exit from the cell cycle. Introduction During adipose conversion of mouse 3T3-Li cells, a discrete period of mitotic divisions separates the precursor adipoblasts from adipocytes. This discrete period of mitotic growth has been called clonal expansion (i). In the standard adipose conversion protocol (2), logarithmically growing Received 4/30/97; revised 8/13/97; accepted 8/i 4/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 1 8 U.S.C. Section 1 734 solely to mdicafe this fact. 1 This work was supported by American Cancer Society Grant BE-i 83 and by a grant from the Jeffress Trust (to R. M. U.). 2 To whom requests for reprints should be addressed, at Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903. Phone: (804) 982-58i 5; Fax: (804) 982-5626; E-mail: rmu4b@virginia.edu. 3T3-Li adipoblasts are propagated to growth arrest at contact inhibition. The cells are held until 48 h postconfluence, defined as day 0, and differentiation is initiated by stimulating the cells with a cocktail of adipogenic hormones, including the glucocorticoid dexamethasone, MIX3 (an inhibitor of cAMP phosphodiesterase), and insulin. In response to the hormones, the cells reenter the cell cycle and clonally expand. Upon removal of the adipogenic hormones after 48 h, on day 2, the cells enter an irreversible growth arrest and begin to express adipocyte-specific genes (3, 4). The synchronous behavior of the 3T3-Li population during this transitional period (5) affords the opportunity to dissect the coupling of cellular growth control to adipocyte differentiation. Each of the individual adipogenic hormones is necessary for optimal differentiation of 3T3-Lis (6). Adipoblasts deprived of dexamethasone or MIX exhibit minimal differentiation and intermediate mitogenic response (6). The undifferentiated cells persisting after suboptimal stimulation are morphologically indistinguishable from day 0 adipoblasts (6); however, their growth properties have not been examined. Presumably, the adipogenic hormones act in concert to both initiate the differentiation program and establish postmitotic growth arrest. Each hormone induces a different subset of proteins. There are changes in the expression of over 300 proteins in response to the addition of the differentiation hormones (7). Of the 100 proteins that increase in expression within 5 h after hormone exposure, the majority increase in response to MIX and insulin, whereas only 1 0 proteins are induced by dexamethasone (7). A limited number of proteins have been characterized as being maximally expressed during the period of clonal expansion, including protein-tyrosine phosphatase HA2 (8), FK506-binding protein (FKBP5i ; Ref. 9), and two members of the CCAAT/enhancer binding protein (C/EBP) family, C/EBPf3 and C/EBPS (i 0). C/EBPI3 continues to be expressed in adipocytes, whereas C/EBP expression is largely confined to the period of clonal expansion (i 0). The accumulation of the two C/EBP isoforms has been established as downstream consequences of distinct adipogenic hormones. Specifically, increases in cAMP mediate C/EBPI3 accumulation, whereas dexamethasone affords increases in C/EBP6 (iO). Dexamethasone regulates differentiation through the activity of adipogenic transcription factors. In the 3T3-Li model system, dexamethasone deprivation during clonal expansion precludes the induction of all three C/EBP isoforms and differentiation, whereas MIX deprivation retards the accumulation of the isoforms and reduces differentiation efficiency 3 The abbreviations used are: MIX, methylisobutylxanthine; PPARy, peroxisome proliferator-activated receptor y; gas, growth arrest specific; gadd, growth-arrest and DNA damage-inducible; FBS, fetal bovine serum; TIFF, tagged image file format. entire -dex DAY 0 1 2 3 4 6 1 2 3 4 6 gas2 C -0-entire GAS2 ....& dcx C 0 “ 0 #{216}#{216} , j: