The data presented here demonstrate that CREB activation is necessary and sufficient to initiate the adipocyte differentiation program. This conclusion is based on the constitutive expression of CREB in 3T3-L1 fibroblasts prior to the induction of adipogenesis and throughout the differentiation process. Furthermore, both CREB phosphorylation and transcriptional activity are rapidly induced in 3T3-L1 fibroblasts by conventional differentiation-inducing agents, and CREB appears to bind to and stimulate transcription from the promoters of several adipocyte-specific genes. Most importantly, we have directly demonstrated that CREB stimulates adipogenesis through our ability to induce adipocyte differentiation with constitutively active VP16-CREB and to completely block the efficacy of normal differentiation-inducing agents with dominant-negative KCREB. Obviously, there are caveats to these conclusions based on studies with VP16-CREB and KCREB. The properties of VP16-CREB may differ significantly form those of wild-type CREB, and KCREB may alter the function of factors other than CREB. However, the strength of our conclusion is founded on complementary results generated with positive and negative forms of CREB that elicit opposing responses. In addition, the ability of the chimeric VP16-KCREB protein to block adipogenesis indicates that our data are not due to indirect or nonspecific effects such as transcriptional squelching. Our conclusion is further supported by the ability of VP16-CREB and KCREB to regulate transcription from well-defined, CRE-containing, adipocyte-specific gene promoters.
The induction of adipogenesis by VP16-CREB alone indicates that CREB activation is sufficient to induce this process, whereas the ability of KCREB to block adipogenesis indicates that CREB activation is a necessary step in adipocyte development. These conclusions are significant because factors previously identified as participants in adipogenesis are only expressed in significant levels after initiation of the differentiation program. Our results suggest that CREB is a primary inducer of adipogenesis and, therefore, a potential target for intercellular signaling mechanisms that recruit the development of new fat cells in hyperplastic obesity. Further, CREB and the signaling systems that impinge on CREB may prove to be targets for therapeutic agents to treat or prevent obesity. Interestingly, preliminary experiments in our laboratory indicate that constitutive overexpression of KCREB in mature adipocytes leads to their dedifferentiation with loss of triacylglycerol vesicles, even in the presence of insulin (data not shown). Unger and colleagues (76
) have recently reported a similar reversal of adipocyte phenotype in normal rats after overexpression of leptin. These studies support the contention that adipocyte development and function can be regulated at various levels, thus opening the door to novel strategies designed to address obesity and related disorders such as insulin resistance.
Our data further confirm the concept that CREB and other ATF-cAMP response element modulator (CREM)-inducible cAMP early repressor (ICER) family members play important roles in multiple cellular activities, most notably proliferation and differentiation. Initial clues to CREB's participation in these activities came from studies showing that several growth factors and other extracellular stimuli activate CREB. We demonstrated that insulin stimulates CREB phosphorylation in 3T3-L1 fibroblasts and adipocytes and HepG2 cells through an ERK1/ERK2 signaling system (34
) and a decrease in nuclear PP2A activity (49
). Greenberg, and colleagues have reported a similar signaling cascade to CREB for nerve growth factor in neuronal cells (27
). Likewise, fibroblast growth factor (60
) and insulin-like growth factor 1 (46
) also stimulates CREB phosphorylation and activity in neuronal cells, but this process appears to be mediated by p38 MAP kinase rather than ERK1/ERK2. CREB and related proteins have also been implicated in the G1
-S transition of the cell cycle in studies showing that cyclin A gene transcription is stimulated by cAMP agonists via CRE sequences in the cyclin A gene promoter (19
In addition to this circumstantial evidence promoting a role for CREB and related factors in cell growth and differentiation, several groups have recently reported direct evidence supporting this hypothesis. For example, Shimomura et al. (56
) have reported that a dominant-negative ATF-1 protein blocks cAMP-induced neurite outgrowth in PC12 cells. Likewise, ectopic expression of a dominant-negative CREB protein in pituitary somatotrophic cells leads to somatotroph hypoplasia and dwarfism in transgenic mice (59
). Targeted expression of a dominant-negative CREB in cardiac myocytes has been shown to produce idiopathic-dilated cardiomyopathy with exaggerated heterogeneity in the myocyte phenotype (21
). Surface antigen receptor activation of B lymphocyte proliferation appears to involve enhanced CREB phosphorylation in response to elevated PKA and PKC activity and downregulation of PP2A (4
), and the expression of dominant-negative CREB in T lymphocytes blocks their proliferation after activation (9
). CREB null transgenic mice exhibit perinatal mortality, reduced corpus callosum and anterior commissures in the brain, decreased thymic cellularity, and impaired T lymphocyte development (52
). cAMP signaling to CREM and ICER via PKA has been shown to play a role in hepatocyte proliferation (53
), and CREB phosphorylation directly inhibits hepatic stellate cell proliferation (31
). Similarly, cAMP-induced ICER IIγ expression blocks the proliferation of either mouse pituitary tumor cells or human choriocarcinoma cells at the G2
-M boundary (48
). Lamas et al (36
) have reported that the CREB inhibitor, ICER, modulates pituitary corticotroph proliferation. In other studies, the tissue-specific extinguisher locus (TSE-1) identified by Fournier and colleagues (11
), which presumably blocks PKA signaling to CREB and other factors, accounts for loss of hepatocyte phenotype markers in hepatoma-fibroblast hybrids. The data presented here extend the multifunctional role of CREB by demonstrating for the first time that activation of this factor is necessary and sufficient to induce a differentiation program by using constitutively active and dominant-negative forms of CREB.
One concern raised by these studies regards the paradoxical role of cAMP signaling in both adipogenesis and lipolysis. Our data are consistent with previous reports demonstrating a key role for cAMP in potentiating adipogenesis (40
). However, other laboratories have shown that β3-adrenergic stimulation of cAMP-PKA signalling increases lipolysis (16
), and targeted knockout of the RIIβ subunit of PKA leads to decreased obesity in mice (17
). These contradictory processes may be reconciled based on different roles for cAMP-PKA signaling between undifferentiated fibroblasts compared to mature adipocytes. Similarly, differentiation of fibroblasts to adipocytes is induced by the transient application of high levels of cAMP mimetics, whereas β3-adrenergic stimulation or RIIβ subunit knockout probably represents protracted increases in cAMP-PKA signaling. Thus, differences in experimental models may account for the seemingly contradictory role of cAMP in adipogenesis and lipolysis. Moreover, it should be remembered that cAMP and PKA regulate numerous intracellular systems and not just CREB and that, more importantly, CREB function can be regulated by a variety of growth factors and not just increases in cAMP. Together, these concepts support a model in which multiple signals may impinge upon CREB to induce adipogenesis in fibroblasts, whereas lipolysis is the result of cAMP-PKA signaling to increase the activity of lipolytic pathways in mature adipocytes. Obviously, this is an area which will require significant investigation to unravel the underlying factors, their roles, and their interactions.
Another question not fully addressed by these studies concerns the target(s) which CREB modulates in order to induce adipogenesis. Our preliminary data indicate that CREB can bind to putative CREs in the promoters of several adipocyte-specific genes. Most the sequences we examined (with the exception of the CEBPδ sequence) have been shown by other groups to interact with nuclear factors and to participate in gene expression in response to cAMP and/or insulin (13
). Furthermore, the genes encoding PEPCK, FABP, and CEBPβ have been shown to be acutely regulated by cAMP or insulin, and the PEPCK and CEBPβ sequences we tested have been shown to confer cAMP and CREB responsiveness on these genes. Certainly, our data are insufficient to permit us to conclude that CREB directly regulates the genes we selected. However, the results provide tantalizing evidence that CREB may regulate certain adipocyte-specific genes, which would support a role for CREB in adipogenesis. We have initiated experiments to directly asses CREB's role in regulating a group of candidate genes, as well as identify other “CREB-regulated, adipocyte-specific” genes via gene microarray analysis.
The binding of CREB to an oligonucleotide probe corresponding to a sequence in the CEBPβ promoter was particularly interesting. As noted before, CEBPβ is expressed very early in adipogenesis and will induce the differentiation of fibroblasts to adipocytes when expressed ectopically (75
). Our data suggest that one mechanims by which CREB may induce adipocyte differentiation is through an ability to stimulate CEBPβ expression, which may be sufficient to induce the entire adipogenic cascade. If true, it should be possible to block CREB-induced adipogenesis by inhibiting CEBPβ expression or activity. Figure B shows that CREB undergoes cyclical increases and decreases in phosphorylation (and presumably in transcriptional activity) during adipogenesis. These results imply that CREB may be crucial at other steps in adipocyte differentiation—from an initial stimulation of CEBPβ expression to the late expression of genes encoding PEPCK, FABP, and FAS.
How does CREB regulate growth in certain cell lines and differentiation in others? One possible mechanism hinges on the availability or accessibility of proliferation-related genes in some cells and tissues versus the accessibility of differentiation-inducing genes and phenotype markers in other cell types. Applying this mechanism to adipogenesis suggests that only differentiation-inducing and/or adipocyte-specific genes rather than proliferation-inducing are accessible to CREB in preadipocytes. Another possible mechanisms focuses on the interactions of CREB with other transcription factors that, in concert, exert proliferative versus differentiation-inducing effects in a cell- or tissue-dependent manner. Interactions between CREB and other transcription factors have been described in several systems, but their role in adipogenesis remains unclear. A number of possible mechanisms may account for CREB's participation in both proliferation and differentiation pathways. It will be interesting to determine which mechanisms are actually functioning in these capacities and to define potential interactions between the mechanisms in the coordinate regulation of these processes.