Reduced expression of AP27 protein, the product of a growth factor-repressible gene, is associated with diminished adipocyte differentiation.

Abstract

We have recently characterized an adipocyte cDNA (clone 5) that is enhanced in expression by environmental and hormonal conditions favoring adipogenic differentiation. Moreover, certain agents including fibroblast growth factor and phorbol 12-myristate 13-acetate (but not epidermal growth factor) markedly inhibit clone 5 gene expression and prevent TA1 cell differentiation. These results led us to propose that a threshold level of the clone 5 gene product (AP27 protein) is required for triggering adipocyte differentiation. We have constructed vectors that direct the synthesis ofclone S antisense RNA to reduce the levels of AP27 in adipogenic cell lines TA1 and 3T3-L1. We show here that when these cells express clone S antisense RNA, they fail to undergo morphological differentiation, whereas adipogenesis is unaffected in cells expressing antisense fi-actin or ferritin heavy-chain RNA. We further show that cells expressing clone S antisense RNA (but not the other antisense RNAs) are unable to induce the expression of characteristic "adipocyte-specific" mRNAs. The level of inhibition of differentiation by clone 5 antisense RNA correlates with decreased levels of AP27 protein. These results provide strong evidence that expression of AP27 is linked to adipogenic differentiation and that AP27 may be a component of an as-yet-uncharacterized signal-transduction pathway required for the triggering of adipocyte differentiation.Adipogenic stem cells, such as 3T3-L1 (1) and TA1 (2), are widely used to study the mechanisms by which hormonal and environmental signals control the switch from a progenitor cell to its fully differentiated counterpart. Such cell lines resemble fibroblasts while growing at low density, but upon reaching confluence the cells stop growing and in time take on the morphological and biochemical characteristics of mature adipocytes (3). Although recent advances have produced insights into the transcriptional mechanisms mediating differentiation-specific gene expression (4,5), there is still a paucity of data relating to the mechanisms by which hormonal and environmental signals are transduced to the elements mediating adipocyte-specific gene expression.Many groups have presented studies in which growth factors were shown to inhibit the differentiation of determined (especially myogenic) stem cell lines (6-8). We have recently reported that the differentiation of TA1 cells is markedly suppressed by fibroblast growth factor (FGF) and the phorbol ester 12-myristate 13-acetate (PMA), in a growthindependent fashion, as reflected by the inability of the cells to undergo morphological differentiation or to accumulate adipocyte-specific RNAs. We also described the characteristics of a specific mRNA, clone 5, the expression of which is suppressed by these same agents in undifferentiated, preconfluent cultures. This property, in addition to its induction by differentiation-promoting factors such as glucocorticoids and high cell density, suggested to us that the putative protein product of this mRNA (refe...

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“…Although the apparent amount of antisense transcript in these cells varied, as evaluated by Northern blots, these levels did not correlate with the apparent amount of syndecan-1 mRNA (Figure 3). This result is consistent with several reports in which transfection with antisense cDNA yields an antisense RNA that has little apparent effect on mRNA abundance, but substantially reduces the level of the translation product (Amini et al, 1986;Wenz et al, 1992;Godson et al, 1993). Several mechanisms for this reduction are possible, including enhanced degradation or decreased transport into the cytoplasm of the senseantisense duplex, or reduced core protein translation due to altered binding of the mRNA to the ribosome or another part of the translational machinery, or a combination of these.…”

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