Overexpression of Raidd cDNA inhibits differentiation of mouse preadipocytes

Abstract

RAIDD (RIP-associated ICH-1 homologous protein with a death domain) is an adaptor molecule that mediates the action of cysteine proteases involved in apoptosis. To study the possibility of a novel system of cell ablation mediated by RAIDD, a preadipocyte cell line (3T3L1) was stably transfected with a plasmid containing the murine Raidd cDNA under the control of the adipocyte specific promoter aP2. Instead of the expected apoptosis, a blockage to differentiation upon hormonal induction was observed as judged by an absence of lipid accumulation, a lack of expression of adipocyte-specific genes and a fibroblastic appearance. Proliferation rate of Raidd-transfected clones remained unaffected. Overexpression of Raidd cDNA in 3T3L1 cell therefore inhibited differentiation, suggesting that Raidd plays a role in controlling differentiation of mouse preadipocytes and, perhaps, in other cell types, in addition to its established role in apoptosis.

Figure 1

Differentiation of 3T3L1 cells into adipocytes upon hormonal induction and lack of differentiation in Raidd‐transfected 3T3L1 cells. (a, b, c) are photographs (100 × magnification) of cells 8 days after hormonal induction to differentiation. (a) shows parental 3T3L1 cells that differentiated normally after hormonal induction. In contrast, cells stably transfected with the murine Raidd cDNA did not undergo adipocyte differentiation in response to hormone treatment [(b) and (c) for clones 2 and 6, respectively]. (d, e, f) show cells stained with Oil Red O, which stains specifically the lipids. Dark staining is observed in the parental 3T3L1 cells (d) that accumulate triglycerides indicating appropriate differentiation into adipocytes. No staining is observed in the Raidd ‐transfected clones [(e, f), for clones 2 and 6, respectively] confirming the blockage of differentiation in these clones.

Figure 2

Raidd expression from the introduced construct and adipsin expression, a marker for differentiation. 5 µg of total RNA from cells 8 days after hormonal induction was DNAse treated, reverse transcribed with oligo (dT)12–18 mer (Gibco BRL) and subjected to PCR with primers (see Material and methods) specific for Raidd transcript from the introduced construct and adipsin. All six Raidd ‐transfected clones (1–6) express Raidd transcript from the construct. Adipsin, a marker of terminally differentiated adipocytes, is expressed only in Raidd ‐transfected clones 1 and 4 that partially differentiated, and in control cells 8 days after hormonal induction (A). No adipsin expression is detected in Raidd ‐clones exhibiting a complete blockage to differentiation (clones, 2, 3, 5, 6) and in control cells before hormonal induction (B).

Figure 3

Raidd overexpression inhibits terminal markers of the adipogenic programme. Ten micrograms of total RNA from two Raidd‐ transfected clones and the parental cells 3T3L1 after different days of the differentiation programme were run on a denaturing agarose gel and northern blot hybridized with probes: aP2, C/EBPα, C/EBPβ and 7S as loading control.

Figure 4

Cell number quantification at different time points of the differentiation programme. Cell number of the parental cells 3T3L1 (empty bar) and one of the clones transfected with Raidd (solid bar) were counted at different points of the differentiation programme, including at confluence (−2) after hormonal induction (+2) and after 6 days of differentiation (+6). Each bar represents the mean ± SD of duplicates.