Microarray analysis of differentiation-specific gene expression during 3T3-L1 adipogenesis

Abstract

During cellular differentiation and development, it is recognized that many complex molecular mechanisms as well as precise patterns of differentially expressed genes occur in directing precursor cells toward a given lineage. Using microarray-based technology, we examined gene expression across the course of 3T3-L1 adipocyte differentiation. Total cellular RNA was isolated at times 0, 2, 8, 16, 24, 48, and 96 h following treatment with either standard hormonal inducers of differentiation; insulin, dexamethasone, isobutylmethylxanthine (IDX), or IDX plus trichostatin A (TsA), a histone deacetylase inhibitor and potent adipogenic inhibitor. cRNA was synthesized from cellular RNA and hybridized to high density Affymetrix MG_U74Av2 microarray gene chips containing 12,488 cDNA/Expressed Sequence Tags (ESTs) probe sets. From the IDX-only treated cells, all probe sets that were either unchanged or differentially expressed less than 2-fold throughout differentiation with respect to time 0 preadipocytes were excluded from further analyses. This selection resulted in a net of 1686 transcripts, 859 were increased in expression, and 827 were decreased in expression at least 2-fold across differentiation. To focus in on genes that were more specific to differentiation, the same analysis was performed on IDX plus TsA-treated non-differentiating cells and all probe sets from the IDX-only group that exhibited similar expression profiles in the non-differentiating TsA-treated group were excluded leaving a total of 1016 transcripts that were regulated only under differentiating conditions. Six hundred and thirty-six of these transcripts were elevated at least 2-fold and 380 exhibited a decrease in expression relative to time 0 preadipocytes. This group of genes was further analyzed using hierarchical clustering and self-organizing maps and resulted in the identification of numerous genes not previously known to be regulated during adipocyte differentiation. Many of these genes may well represent novel adipogenic mediators and markers of adipogenesis.

Introduction

Obesity represents a tremendous international health problem and serves as a significant risk factor for many diseases such as diabetes, cancer, heart disease and hypertension MacDougald and Lane, 1995, Cowherd et al., 1999. Related to obesity, many recent investigations have focused on the differentiation of adipocytes, the predominant cell present within adipose tissue. Many of these studies have utilized in vitro models of adipocyte differentiation, and one of the most well characterized and widely used has been the 3T3-L1 preadipocyte cell line MacDougald and Lane, 1995, Cowherd et al., 1999. Adipogenic differentiation of these cells is accomplished over a 4-day hormonally induced treatment regimen containing insulin, dexamethasone, and isobutylmethylxanthine, designated IDX (MacDougald and Lane, 1995, Cowherd et al., 1999). During early differentiation (∼24 h), the cells reenter the cell cycle and following one to two mitotic divisions, cells permanently withdraw from cell cycle and undergo terminal differentiation MacDougald and Lane, 1995, May et al., 2001, Prince et al., 2002.

The process of cellular differentiation represents a remarkably coordinated regulation of gene expression that directs multipotential stem cell precursors down various lineages into fully mature and functionally distinct cell types. During adipocyte differentiation, numerous investigators have demonstrated that many genes are regulated in a differentiation-dependent manner. Some are only briefly expressed, such as the retinoblastoma genes and the E2F family of transcription factors that are involved in regulating cell cycle (Richon et al., 1997). Several adipogenic transcription factors have been well described, like the peroxisome proliferator activated receptor-γ (PPARγ) and the CCAAT-enhancer binding protein-α (C/EBPα MacDougald and Lane, 1995). In addition, as preadipocytes differentiate into fully mature adipocytes, they begin to express later markers of differentiation that include the whole cadre of genes associated with lipid transport and metabolism (Cowherd et al., 1999).

In what currently is becoming known as the post-genomic era, many new technologies and methodologies are being developed to take advantage of much of our recent progress in genome sciences. A particularly useful technology has been the development of high density cDNA/oligonucleotide microarrays capable of screening thousands of known genes and expressed sequence tags (ESTs) simultaneously Lockhart et al., 1996, Pietu et al., 1996. RNA, easily obtainable, is the typical input for these microarrays, and this technology allows the simultaneous examination of virtually every transcript expressed in a cell or tissue at any given time from which RNA can be isolated. It represents a particularly powerful tool to examine differentiation by being able to compare gene expression across time and under different experimental conditions. Previously, we utilized a microarray-based approach to analyze changes in global gene expression during the first 24 h of adipocyte differentiation and this along with other studies have established microarray technology as a valid and useful tool in the study of cell differentiation Guo and Liao, 2000, Burton et al., 2002, Ross et al., 2002. The application of microarray technology to differentiation models will not substitute for careful functional analyses, however it represents an ideal approach to identify candidate genes important to differentiation.

The current study examines the complete time course of 3T3-L1 adipocyte differentiation, however, four time points were included within the first 24 h in an attempt to identify early changes in mRNA expression that might otherwise be missed. The first 24 h represent a critical time during 3T3-L1 adipogenesis. For example, this is the time frame for clonal expansion or cell cycle reentry, a process considered requisite to adipogenesis (MacDougald and Lane, 1995). It is during this time that tumor necrosis factor α (TNFα), a potent adipogenic inhibitor, has been demonstrated to exert an inhibitory effect only if administered within the first 24 h (Lyle et al., 1998). Two members of the retinoblastoma (Rb) tumor suppressor family, p130 and p107, are dramatically regulated during the first 24 h of differentiation and altering the differentiation-specific expression of these proteins inhibits differentiation May et al., 2001, Prince et al., 2002. Day 2 (48 h) was also included in this study based on known expression patterns for the adipogenic transcription factors C/EBPα and PPARγ. Studies have shown that induction of C/EBPα expression in 3T3-L1 cells will trigger differentiation even in the absence of treatment with IDX hormone cocktail (Lin and Lane, 1994). Furthermore, C/EBPα antisense loss of function experiments have demonstrated that knocking out the C/EBPα protein prevents terminal differentiation of 3T3-L1 cells (Lin and Lane, 1992). PPARγ is a potent inducer of adipogenesis and many genes such as lipoprotein lipase expressed during mid and late differentiation contain PPAR response elements within their 5′-flanking promoters (Schoonjans et al., 1996). In the present study we utilize microarray technology to analyze changes in global gene expression at seven key time points across differentiation.

Trichostatin A (TsA) is an inhibitor of histone deacetylase (HDAC) and a potent inhibitor of 3T3-L1 adipocyte differentiation, though the mechanism by which TsA inhibits adipocyte differentiation has not yet been elucidated (Classon et al., 2000). Members of the Rb family of tumor suppressors (pRb, p107, and p130) inhibit progression through the cell cycle by binding to and inhibiting the activity of E2F proteins, a family of transcription factors known to be important in the regulation cell cycle progression (Richon et al., 1997). During cell cycle reentry, Rb proteins are phosphorylated by cyclin-dependent kinases (CDKs). Phosphorylated Rb is incapable of binding E2F and therefore, is unable to inhibit E2F-mediated cell cycle gene expression. HDACs are generally associated with gene repression and it has been reported that interactions between HDACs and all three members of the Rb family may be involved in repressing E2F-mediated gene transcription, therefore providing a potential mechanism through which HDAC inhibition by TsA leads to a block in adipocyte differentiation (Zhang and Dean, 2001).

In the current study, microarray analysis was performed in duplicate sets of experiments on differentiating 3T3-L1 cells using the Affymetrix murine MG_U74A containing 12,488 probe sets for both known genes and ESTs. Time points spanning the entire course of IDX-stimulated adipocyte differentiation were chosen for this study and included 0, 2, 8, 16, 24, 48 and 96 h. All probe sets not exhibiting a 2-fold or greater change in expression levels were excluded. The probe sets remaining after this selection include many transcripts that may be regulated in response to the IDX hormonal treatment only and not necessarily related to the differentiation process. Therefore, an identical analysis was performed on differentiation-inhibited cells stimulated with IDX in the presence of TsA. Elimination of genes that were similarly regulated in both experiments allowed the generation of a data set of transcripts that were differentially expressed only in differentiating cells. Here, we report the analysis of these genes detected specifically in differentiating cells that exhibited a 2-fold or greater change in expression levels during adipogenesis.