To investigate the role of C/EBP family members during adipocyte differentiation in vivo, mice have been generated lacking the C/EBPbeta and/or C/EBPdelta by gene targeting. Approximately 85% of C/EBPbeta(-/-)·delta(-/-) mice die at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contains many lipid droplets, whereas C/EBPbeta(-/-)·delta(-/-) mice do not accumulate droplets. The epidydimal fat pad weight of surviving adult C/EBPbeta(-/-)·delta(-/-) mice is significantly reduced, when compared with wild-type mice. However, these adipose tissues in C/EBPbeta(-/-)·delta(-/-) mice exhibit normal expression of C/EBPalpha and PPARgamma, despite impaired adipogenesis. These results demonstrate that C/EBPbeta and C/EBPdelta have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPalpha and PPARgamma does not always require C/EBPbeta and C/EBPdelta, but co-expression of C/EBPalpha and PPARgamma is not sufficient for complete adipocyte differentiation in the absence of C/EBPbeta and C/EBPdelta (Tanaka, 1997).

The C/EBP family of transcription factors is involved in the establishment of the characteristic phenotype of the brown adipocyte. The uncoupling protein gene, responsible for brown fat thermogenesis, is a target for C/EBP-dependent transcriptional regulation. Primary brown adipocytes differentiated in culture were transiently transfected with plasmids containing different extensions of the 5'-flanking region of the rat uncoupling protein gene placed upstream of the bacterial chloramphenicol acetyltransferase reporter gene. Co-transfection into primary brown adipocytes of expression vectors [for CCAAT/enhancer binding protein (C/EBP) alpha and C/EBP beta] trans-activate the rat uncoupling protein gene promoter due to sequences in the 5' proximal region. There are two C/EBP binding sites at positions -457/-440 and -335/-318 that interact with purified C/EBP beta as well as with C/EBP proteins present in brown fat or liver nuclear extracts (Yubero, 1994).

Treatment of 3T3-L1 adipocytes with insulin (IC50 ~200 pM insulin) or insulin-like growth factor-1, stimulates dephosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor involved in preadipocyte differentiation. Insulin appears to dephosphorylate one site within p30C/EBPalpha and an additional site within p42C/EBPalpha. Consistent with insulin causing dephosphorylation of C/EBPalpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPalpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBPalpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPalpha in the absence of insulin. Dephosphorylation of C/EBPalpha by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBPalpha. The dephosphorylation of C/EBPalpha by insulin, in conjunction with the insulin-dependent decline in C/EBPalpha mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBPalpha. However, the repression of C/EBPalpha mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway, without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBPalpha and GLUT4 mRNAs by insulin. A decline in C/EBPalpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPbeta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors (Hemati, 1997).

Terminal differentiation of stem cells is characterized by cessation of cell proliferation as well as changes in cell morphology associated with the differentiated state. For adipocyte differentiation, independent lines of evidence show that certain transcription factors are essential: peroxisome proliferator activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha (C/EBPalpha), as well as the tumor suppressor retinoblastoma (Rb) protein. How these proteins promote adipocyte conversion and how they function cooperatively during the differentiation process remain unclear. Retinoic acid (RA) inhibition of adipogenesis was used to investigate these issues. RA blocks adipogenesis of 3T3-L1 cells induced to differentiate by ectopic expression of PPARgamma and C/EBPalpha independently or together. However, under these circumstances RA is only effective at preventing adipogenesis when added prior to confluence, suggesting that factors involved in regulation of the cell cycle might play a role in establishing the commitment state of adipogenesis that is insensitive to RA. During differentiation of wild type 3T3 L1 preadipocytes, Rb protein is hyperphosphorylated early in adipogenesis, corresponding to previously quiescent cells re-entering the cell cycle, and later becomes hypophosphorylated. The data suggest that permanent exit from the cell cycle establishes the irreversible commitment to adipocyte differentiation, together with the coexpression of PPARgamma and C/EBPalpha (Shao, 1997).

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. C/EBP alpha, as well as another nuclear factor present in adipocytes, binds to the proximal region of the transcriptional start site. Mutation of this site blocks transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter (Hwang, 1996).

Mice homozygous for the targeted deletion of the c/ebp alpha gene do not store hepatic glycogen; they die from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA is 50 to 70 percent of normal. There is also a delay in the transcriptional induction of the genes for two gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase). The hepatocytes and adipocytes of the mutant mice fail to accumulate lipid and there is a reduction in the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates (Wang, 1995).

C/EBP alpha is a necessary factor for follicular development in the rat ovary. Ovaries treated with antisense oligonucleotides to C/EBP alpha exhibit an altered morphology. Ovary exposed to antisense oligonucleotides contained few corpora lutea as a demonstration of a reduced number of ovulations. This suggests that antisense treatement interfers with follicular development resulting in a failure to ovulate in response to an ovulatory dose of human chorionic gonadotropin. C/EBP alpha is involved in the transition of the small antral follicle to a more differentiated stage, i. e., the preovulatory stage, making the follicle capable of responding to the ovulatory surge of Lactogenic hormone (Piontkewitz, 1996).

The gene for phosphoenolpyruvate carboxykinase (PEPCK), a target of CCAAT/enhancer-binding protein-alpha (C/EBPalpha) and -beta (C/EBPbeta), begins to be expressed in the liver at birth. Mice homozygous for a deletion in the gene for CEBPalpha (C/EBPalpha-/- mice) die shortly after birth of hypoglycemia, with no detectable hepatic PEPCK mRNA and negligible hepatic glycogen stores. Half of the mice homozygous for a deletion in the gene for CEBPbeta (C/EBPbeta-/- mice) have normal glucose homeostasis (phenotype A), and the other half die at birth of hypoglycemia due to a failure to express the gene for PEPCK and to mobilize hepatic glycogen (phenotype B). Insulin deficiency induces C/EBPalpha and PEPCK gene transcription in the livers of 19-day fetal rats, whereas dibutyryl cyclic AMP (Bt2cAMP) increases the expression of the gene for C/EBPbeta and causes a transient burst of PEPCK mRNA. Bt2cAMP induces PEPCK mRNA in the livers of fetal C/EBPalpha-/- mice, but at only 20% of the level of control animals; however, there is no induction of PEPCK mRNA if the cyclic nucleotide is injected into C/EBPalpha-/- mice immediately after delivery. The expression of the gene for C/EBPbeta is markedly induced in the livers of C/EBPalpha-/- mice within 2 h after the administration of Bt2cAMP. C/EBPbeta-/- mice injected at 20 days of fetal life with Bt2cAMP have a normal pattern of induction of hepatic PEPCK mRNA. In C/EBPbeta-/- phenotype B mice, the administration of Bt2cAMP immediately after delivery induces PEPCK mRNA, causes the mobilization of hepatic glycogen, and maintains normal glucose homeostasis for up to 4 h (duration of the experiment). It is concluded that C/EBPalpha is required for the cAMP induction of PEPCK gene expression in the liver and that C/EBPbeta can compensate for the loss of C/EBPalpha, if its concentration is induced to appropriate levels (Croniger, 1997).

The beta(3)-adrenergic receptor [beta(3)AR] is expressed predominantly in adipocytes, and it plays a major role in regulating lipolysis and adaptive thermogenesis. Its expression in a variety of adipocyte cell models is preceded by the appearance of CCAAT/enhancer-binding protein alpha (C/EBP alpha), which has been shown to regulate a number of other adipocyte-specific genes. Importantly, it has been demonstrated that several adipocyte cell lines that fail to express C/EBP alpha exhibit reduced insulin sensitivity, despite an apparent adipogenic phenotype. Transcription and function of the beta(3)AR correlates with C/EBP alpha expression in these adipocyte models. A 5.13-kilobase pair fragment of the mouse beta(3)AR promoter was isolated and sequenced. This fragment conferred a 50-fold increase in luciferase reporter gene expression in adipocytes. Two putative C/EBP binding sites exist at -3306 to -3298 and at -1462 to -1454, but only the more distal site is functional. Oligonucleotides corresponding to both the wild-type and mutated -3306 element were inserted upstream of a thymidine kinase luciferase construct. When cotransfected in fibroblasts with a C/EBP alpha expression vector, reporter gene expression increased 3-fold only in the wild-type constructs. The same mutation, when placed into the intact 5.13-kilobase pair promoter, reduced promoter activity in adipocytes from 50-fold to <10-fold. Electrophoretic mobility shift analysis demonstrated that the site at -3306 generated a specific protein-oligonucleotide complex that was supershifted by C/EBP alpha antibody, while a probe corresponding to a putative site at -1462 did not. These results define C/EBP alpha as a key transcriptional regulator of the mouse beta(3)AR gene during adipogenesis (Dixon, 2001).

The transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is expressed at high levels in liver and adipose tissue. Cell culture studies show that C/EBPalpha is sufficient to trigger differentiation of preadipocytes into mature adipocytes, suggesting a central role for C/EBPalpha in the development of adipose tissue. C/EBPalpha knockout mice die within 7-12 h after birth. Defective gluconeogenesis of the liver and subsequent hypoglycemia contribute to the early death of these animals. This short life span impairs investigation of the development of adipose tissue in these mice. To improve the survival of C/EBPalpha-/- animals, a transgenic line was generated that expresses C/EBPalpha under the control of the albumin enhancer/promoter. This line was bred into the knockout strain to generate animals that express C/EBPalpha in the liver but in no other tissue. The presence of the transgene improved survival of C/EBPalpha-/- animals almost 3-fold. Transgenic C/EBPalpha-/- animals at 7 days of age show an absence of s.c., perirenal, and epididymal white fat despite excess lipid substrate in the serum, whereas brown adipose tissue is somewhat hypertrophied and shows minimal biochemical alterations. Interestingly, mammary gland fat tissue is present and exhibits normal morphology. The absence of white adipose tissue in many depots in the presence of high serum lipid levels shows that C/EBPalpha is required for the in vivo development of this tissue. In contrast, brown adipose tissue differentiation is independent of C/EBPalpha expression. The presence of lipid in brown adipose tissue serves as an internal nutritional control, indicating that neither nutritional intake nor lipoprotein composition is likely responsible for the absence of white fat (Linhart, 2001).

PPARgamma and C/EBPalpha are critical transcription factors in adipogenesis, but the precise role of these proteins has been difficult to ascertain because they positively regulate each other's expression. Questions remain about whether these factors operate independently in separate, parallel pathways of differentiation, or whether a single pathway exists. PPARgamma can promote adipogenesis in C/EBPalpha-deficient cells, but the converse has not been tested. An immortalized line of fibroblasts lacking PPARgamma was created, which was used to show that C/EBPalpha has no ability to promote adipogenesis in the absence of PPARgamma. These results indicate that C/EBPalpha and PPARgamma participate in a single pathway of fat cell development with PPARgamma being the proximal effector of adipogenesis (Rosen, 2002).

Brown fat differentiation in mice is fully achieved in fetuses at term and entails the acquisition of not only adipogenic but also thermogenic and oxidative mitochondrial capacities. The present study of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein alpha (C/EBPalpha-null mice) demonstrates that C/EBPalpha is essential for all of these processes. Developing brown fat from C/EBPalpha-null mice show a lack of uncoupling protein-1 expression, impaired adipogenesis, and reduced size and number of mitochondria per cell when compared with wild-type mice. Furthermore, immature mitochondrial morphology was found in brown fat, but not in liver or heart, from C/EBPalpha-null mice. Concordantly, expression of both nuclear and mitochondrial genome-encoded genes for mitochondrial proteins was reduced in C/EBPalpha-null brown fat, although expression of mitochondrial rRNA and mitochondrial DNA content were unaltered. Expression of nuclear respiratory factor-2, thyroid hormone nuclear receptors, and peroxisome proliferator-activated receptor gamma coactivator-1, was delayed in C/EBPalpha-null brown fat. Iodothyronine 5'-deiodinase activity and thyroid hormone content were also reduced in brown fat from C/EBPalpha-null mice, indicating for the first time a crucial role for C/EBPalpha in controlling thyroid status in developing brown fat; thyroid status may contribute to impaired mitochondrial biogenesis and cell differentiation. When survival of C/EBPalpha-null mice was achieved by transgenically expressing C/EBPalpha only in the liver, a substantial recovery in brown fat differentiation was found by day 7 of postnatal age, which is associated with a compensatory overexpression of C/EBPdelta and C/EBPbeta (Carmona, 2002).

CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPß, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPß, generating the ß/ß alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, ß/ß mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of ß/ß mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The ß/ß allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galpha_s) in WAT. Galpha_s, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of ß/ß mice, and effectively diminished the stored lipid pool (Chiu, 2004).

The GTPase cycle between Gßgamma-dimer and Galpha subunit in G protein-coupled receptor signaling is well established. However, a number of effectors have been shown to be regulated by Gßgamma-dimer via direct protein interaction. These effectors include several adenylyl cyclase (AC) isoforms, voltage-gated Ca2+ channels, and muscarinic K+ channels. For example, Gßgamma-dimer is a stimulator of Type II and Type IV AC isoforms, but an inhibitor of Type I AC. The Gßgamma-dimer may also exert its cellular effect indirectly by interaction with other proteins, such as calmodulin and mitogen-activated protein kinase. In the WAT fat cells of ß/ß mice, it is unlikely that the effect of Galphas was in the main associated with the activation of ß-ARs, because ß-AR expression was markedly reduced in the WAT of ß/ß mice, possibly due to the lack of C/EBPalpha. One possibility is that the elevated levels of Galphas effectively compete with other Galpha subunits for the free form of Gßgamma-dimer, and subsequently prevent the Gßgamma-dimer and/or activation of other Galpha subunits from exerting their cellular effects that may include inhibiting AC activity (Chiu, 2004).

Adipocytes express all three subtypes of ß-AR (ß1, ß2, and ß3), of which each is coupled to the Galphas-cAMP pathway. Thus, in adipocytes, especially of brown adipose tissue, Galphas role in mediating signaling from the ß-AR activation to elicit lipolysis and thermogenesis has been well documented. An inhibitory role of Galphas in adipogenesis of 3T3-L1 cells has been suggested on the basis of the observations that Galphas expression declines markedly within 24 h of adipogenic induction, and that antisense Galphas oligodeoxynucleotides accelerate adipocyte differentiation. In postdifferentiation, however, the regulatory role of Galphas in adipocyte constitution is not yet defined. Interestingly, C/EBP gene replacement increased and maintained the Galphas expression specifically at the state of postadipogenic induction when the preadipocytes isolated from WAT of ß/ß mice were adipogenically induced. This was corroborated by the WAT-specific increase in Galphas expression in ß/ß mice. The increases in mitochondrial biogenesis and metabolic activity in lipid-rich 3T3-L1 cells after overexpression of Galphas at the postadipogenic induction stage further suggests an active role of Galphas in defining adipocyte composition during adipocyte differentiation (Chiu, 2004).

In conclusion, this study indicates that the ß/ß allele changes the metabolic state of WAT adipocytes from energy storage to energy dissipation, possibly via an increased expression of Galphas. The increase in energy oxidation alone in fat cells appears to be able to reverse both genetic and dietary obesities. Regardless of the cause of obesity, all forms of obesity lead to an accumulation of massive quantities of fat in WAT. Thus, increasing the oxidative activity of WAT might be an effective treatment for obesity. Moreover, as overexpression alone of Galphas effectively increased mitochondrial biogenesis and prevented fat accumulation in lipid-rich cells, Galphas might play an active role in programming the lipid-rich cells to be as efficient energy oxidizers as the adipocytes of BAT in which Galphas is highly expressed (Chiu, 2004).

Stimulation of adipogenesis in mouse preadipocytes requires C/EBPbeta as well as activation of the MEK/extracellular signal-regulated kinase (ERK) signaling pathway. Phosphorylation of C/EBPbeta at a consensus ERK/glycogen synthase kinase 3 (GSK3) site regulates adiponectin gene expression during the C/EBPbeta-facilitated differentiation of mouse fibroblasts into adipocytes. Exposure of 3T3-L1 preadipocytes to insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) leads to the phosphorylation of C/EBPbeta at threonine 188. Pretreating the cells with a MEK1-specific inhibitor (U0126) significantly attenuates this activity. Similarly, these effectors activate the phosphorylation of T188 within an ectopic C/EBPbeta overexpressed in Swiss mouse fibroblasts, and this event involves both MEK1 and GSK3 activity. Expression of C/EBPbeta (p34kD LAP isoform) in Swiss mouse fibroblasts exposed to DEX, MIX, and insulin induces expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and some adiponectin but it does not activate expression of FABP4/aP2. In fact, complete conversion of these fibroblasts into lipid-laden adipocytes, which includes activation of FABP4 and adiponectin expression, requires their exposure to a potent PPARgamma ligand such as troglitazone. Expression of a mutant C/EBPbeta in which threonine 188 has been modified to alanine (C/EBPbeta T188A) can induce PPARgamma production in the mouse fibroblasts, but it is incapable of stimulating adiponectin expression in the absence or presence of troglitazone. Interestingly, replacement of T188 with aspartic acid creates a C/EBPbeta molecule (C/EBPbeta T188D) that possesses adipogenic activity similar to that of the wild-type molecule. The absence of adiponectin expression correlates with a reduced amount of C/EBPalpha in the adipocytes expressing the T188A mutant, suggesting that C/EBPalpha is required for expression of adiponectin. In fact, ectopic expression of PPARgamma in C/EBPalpha-deficient fibroblasts (NIH 3T3 cells) produces a modest amount of adiponectin, whereas expression of both PPARgamma and C/EBPalpha in NIH 3T3 cells facilitates production of abundant quantities of adiponectin. These data demonstrate that phosphorylation of C/EBPbeta at a consensus ERK/GSK3 site is required for both C/EBPalpha and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes (Park, 2004).

Hormonal induction of growth-arrested 3T3-L1 preadipocytes rapidly activates expression of CCAAT/enhancer-binding protein (C/EBP) beta. Acquisition of DNA-binding activity by C/EBPbeta, however, is delayed until the cells synchronously enter the S phase of mitotic clonal expansion (MCE). After MCE, C/EBPbeta activates expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma, which then transcriptionally activate genes that give rise to the adipocyte phenotype. Dominant negative C/EBP, A-C/EBP, which possesses a leucine zipper but lacks functional DNA-binding and transactivation domains, forms stable inactive heterodimers with C/EBPbeta in vitro. Infection of 3T3-L1 preadipocytes with an adenovirus A-C/EBP expression vector interferes with C/EBPbeta function after induction of differentiation. A-C/EBP inhibits events associated with hormone-induced entry of S-phase of the cell cycle, including the turnover of p27/Kip1, a key cyclin-dependent kinase inhibitor, expression of cyclin A and cyclin-dependent kinase 2, DNA replication, MCE, and, subsequently, adipogenesis. Although A-C/EBP blocks cell proliferation associated with MCE, it does not inhibit normal proliferation of 3T3-L1 preadipocytes. Immunofluorescent staining of C/EBPbeta reveals that A-C/EBP prevents the normal punctate nuclear staining of centromeres, an indicator of C/EBPbeta binding to C/EBP regulatory elements in centromeric satellite DNA. The inhibitory effects of A-C/EBP appear to be due primarily to interference with nuclear import of C/EBPbeta caused by obscuring its nuclear localization signal. These findings show that both MCE and adipogenesis are dependent on C/EBPbeta (Zhang, 2004).

Zinc finger proteins constitute the largest family of transcription regulators in eukaryotes. These factors are involved in diverse processes in many tissues, including development and differentiation. This study reports the characterization of the zinc finger protein ZNF638 as a novel regulator of adipogenesis. ZNF638 is induced early during adipocyte differentiation. Ectopic expression of ZNF638 increases adipogenesis in vitro, while its knock down inhibits differentiation and decreases the expression of adipocyte specific genes. ZNF638 physically interacts and transcriptionally cooperates with C/EBPβ and C/EBPδ. This interaction leads to the expression of PPARγ, the key regulator of adipocyte differentiation. In summary, ZNF638 is a novel, and early, regulator of adipogenesis that works as a transcriptional cofactor of C/EBPs (Meruvu, 2011).

Adipocyte differentiation is an important component of obesity, but how hormonal cues mediate adipocyte differentiation remains elusive. BMP stimulates in vitro adipocyte differentiation, but the role of BMP in adipogenesis in vivo is unknown. Drosophila Schnurri (Shn) is required for the signaling of Decapentaplegic, a Drosophila BMP homolog, via interaction with the Mad/Medea transcription factors. Vertebrates have three Shn orthologs, Shn-1, -2, and -3. This study reports that Shn-2^-/- mice have reduced white adipose tissue and that Shn-2^-/- mouse embryonic fibroblasts cannot efficiently differentiate into adipocytes in vitro. Shn-2 enters the nucleus upon BMP-2 stimulation and, in cooperation with Smad1/4 and C/EBPα, induces the expression of PPARγ2, a key transcription factor for adipocyte differentiation. Shn-2 directly interacts with both Smad1/4 and C/EBPα on the PPARγ2 promoter. These results indicate that Shn-2-mediated BMP signaling has a critical role in adipogenesis (Jin, 2006).

BMP-2 induces PPARγ expression and adipogenesis in C3H10T1/2 cells. The effects of BMP-2 on PPARγ2 promoter activity was analyzed using a PPARγ2 promoter-driven luciferase gene. Wild-type MEFs were transfected with the PPARγ2-Luc reporter, and adipocyte differentiation was induced in the presence or absence of BMP-2. The luciferase levels of wild-type cells increased 73% in the presence of BMP-2, whereas the luciferase levels of Shn-2^-/- cells were not affected by BMP-2 treatment. Thus, the PPARγ2 promoter is weakly responsive to BMP-2, and Shn-2 is required for this BMP responsiveness. The low degree of induction by BMP-2 could be due to an imbalance among the transcription factors and the promoter molecule in transfected cells (Jin, 2006).

To further examine the BMP responsiveness of the PPARγ2 promoter and the role of Shn-2, luciferase reporter assays were performed using wild-type MEFs transfected with the PPARγ2-Luc reporter and various combinations of expression plasmids for Smad1/4 and Shn-2. The PPARγ2 promoter contains C/EBP binding sites and its activity is enhanced by C/EBPα and C/EBPδ, and, therefore, the C/EBPα expression plasmid was also used. Without Smad1/4 or C/EBPα, the BMP-2-induced expression of luciferase was not observed, whereas BMP-2 enhanced luciferase expression about 2-fold in the presence of Smad1/4 and C/EBPα. When Smad1/4, C/EBPα, and Shn-2 were coexpressed together, higher BMP-2 responsiveness (3.6-fold) was observed. These results may support the speculation that the appropriate balance of these factors and the PPARγ2 promoter molecule is needed for BMP responsiveness. When Shn-2^-/- MEFs were used for similar experiments, BMP-2 enhanced luciferase expression only about 50% in the presence of Smad1/4 and C/EBPα. Exogenous expression of Shn-2 in the mutant cells significantly restored the BMP-2 responsiveness of the PPARγ2 promoter (4.5-fold). These results suggest that Shn-2, Smad1/4, and C/EBPα synergistically mediate the BMP-induced transactivation of the PPARγ2 promoter (Jin, 2006).

Smad3/4 bind to the 5'-AGAC-3' sequence, while Smad1 binds to GC-rich sequences. The mouse and human PPARγ2 promoter regions contain six AGAC sequences but not the GC-rich sequence. The AGAC sequence was also found at ten sites in the 1.2 kb promoter region of the mouse PPARγ1 gene. Among these six putative Smad binding sites in the mouse PPARγ2 promoter, four sites (sites 1, 2, 4, and 6) are conserved in the human PPARγ2 promoter. Mutant mouse PPARγ2-Luc reporters in which the AGAC sites were mutated, and the level of activation of the reporters by Shn-2, Smad1/4, ALK3QD, and C/EBPα was examined. The results indicate that three sites in the upstream region of the promoter (sites 1-3) are required for synergistic activation by these factors. Mutation of any of these three sites significantly reduced activation by Shn-2, Smad1/4, ALK3QD, and C/EBPα. The human PPARγ2 promoter lacks site 3 but has another Smad site further upstream of site 1. The presence of three Smad sites in this region of the mouse and human PPARγ2 promoters may support formation of a Smad1/4-Shn-2-C/EBPα complex to synergistically activate transcription (Jin, 2006).

Vertebrate Shn was originally identified as NF-κB site binding proteins, and the metal finger regions of Drosophila and Xenopus Shn recognize these specific DNA sequences. No NF-κB recognition sequence was found in the PPARγ2 promoter, but one sequence (5'-TCCCACCTCTCCC-3') at -94 to -82 partially resembles the Xenopus Shn binding sequence. However, mutation of this site did not affect the synergistic activation of the PPARγ2 promoter by Shn-2, Smad1/4, ALK3QD, and C/EBPα (Jin, 2006).

To examine whether Shn-2 directly binds to the PPARγ2 promoter, a DNA precipitation assay was performed. FLAG-Shn-2 was expressed in 293T cells, immunoprecipitated by anti-FLAG antibody, and eluted from the immunocomplex using FLAG peptide. The purified Shn-2 protein was mixed with ³²P-labeled PPARγ2 promoter fragments and precipitated with anti-Shn-2 antibody. The PPARγ2 promoter fragment was not detected in the immunocomplex. These results suggest that Shn-2 does not directly bind and is recruited by Smad proteins to the PPARγ2 promoter (Jin, 2006).

To investigate the interaction between Shn-2 and Smad1/4, coimmunoprecipitation assays of the exogenously expressed proteins were performed. 293T cells were cotransfected with plasmids to express FLAG-Shn-2, Myc-Smad1, and HA-Smad4, and lysates from transfected cells were immunoprecipitated with an anti-FLAG antibody. Myc-Smad1 and HA-Smad4 were coprecipitated with FLAG-Shn-2. When HA-Smad4 was deleted from this combination, FLAG-Shn-2 coprecipitated lesser amounts of Myc-Smad1. When Myc-Smad1 was deleted, HA-Smad4 was not coprecipitated with FLAG-Shn-2. These results suggest that Shn-2 interacts with the hetero-oligomers of Smad1 and Smad4 (Jin, 2006).

To determine which region of Shn-2 protein is responsible for interaction with Smad1, GST pull-down assays were performed. Two in vitro-translated Shn-2 fragments containing either the N- or C-terminal metal fingers (N1 and C1) bound to a GST-Smad1 resin, whereas the two fragments containing the central region of Shn-2 (HS and CP) exhibited only background and minor binding, respectively. Deletion of the metal finger regions from N1 and C1 abrogated the interaction with Smad1, suggesting that both metal finger regions are important for interactions with Smad1 (Jin, 2006).

The present study demonstrates that Shn-2 enters the nucleus upon BMP stimulation and plays an important role in adipocyte differentiation. The current study strongly suggests that BMP has a critical role in vivo. Upon BMP stimulation, Shn-2 is recruited to the PPARγ2 promoter via an interaction with Smad1. This is the first demonstration that Shn plays a role in vertebrate BMP/TGF-β/activin signaling. Shn-2 is required for efficient transcription of PPARγ2, possibly as a scaffold protein to form a ternary complex with Smad1/4 and C/EBPα. Interestingly, Evi-1, which is also a large protein containing two regions of metal fingers like Shn-2, interacts with and represses TGF-β/BMP-activated transcription through Smad proteins. Following TGF-β stimulation, Evi-1 and the associated corepressor CtBP are recruited to the target promoter. Thus, Shn-2 and Evi-1 interact with Smad proteins via their metal fingers and may stimulate and repress transcription by recruiting coactivator and corepressor, respectively (Jin, 2006).

Although vertebrate Shn proteins were originally identified as the NF-κB site binding proteins, the present study indicates that Shn-2 is recruited to the PPARγ2 promoter via an interaction with Smad1 and C/EBPα. This is similar to the recent report that Drosophila Shn forms a complex with Mad/Medea on the silencer element of the brinker (brk) gene to mediate Dpp-dependent brk gene silencing. The brinker silencer element contains three 5'-AGAC-3' sequences and two GC-rich sequences between them, to which Medea and Mad bind, respectively. However, the GC-rich sequence was not found between three 5'-AGAC-3' sequences in the PPARγ2 promoter. Therefore, more work is required to understand whether Smad1 in the Smad1/4 hetero-oligomers directly recognizes the DNA sequence in the PPARγ2 promoter. Interaction of Shn-2 not only with Smad1/4 but also with C/EBPα may support the idea that Shn-2 serves as a scaffold protein to form a ternary complex with various transcription factors to synergistically activate transcription. In fact, Shn-3 was reported to interact with c-Jun to activate IL-2 gene transcription (Jin, 2006).

Adipogenesis in vitro follows a highly ordered and well-characterized temporal sequence. In cultured cell models, initial growth arrest is induced by the addition of a prodifferentiative hormonal regimen and is followed by one or two additional rounds of cell division (clonal expansion). This process ceases upon induction of PPARγ2 and C/EBPα, which is concomitant with permanent growth arrest followed by expression of the fully differentiated phenotype. E2F1 induces PPARγ2 transcription during clonal expansion, whereas E2F4 represses PPARγ2 expression during terminal adipocyte differentiation. Interaction between Smad and E2F proteins has been shown for the Smad3-E2F4/5 complex mediating TGF-β-induced repression of c-myc. Therefore, Smad1/4-Shn-2 may also participate in E2F-dependent transcriptional regulation of PPARγ2 by directly interacting with E2F1/4. IFNγ decreases the expression of PPARγ2 in preadipocytes, but the mechanism remains to be elucidated. IFNγ induces the expression of Smad7, which prevents TGF-β receptor-mediated Smad3 phosphorylation. IFNγ may suppress PPARγ2 transcription by inducing Smad6, which then prevents BMP receptor-mediated Smad1 phosphorylation. FoxO1 is also known to regulate adipocyte differentiation. FoxO1 is induced in the early stages of adipocyte differentiation, and prevents adipose differentiation by upregulating multiple genes, including cell cycle inhibitors. Insulin leads to nuclear exclusion of FoxO1 and stimulates adipocyte differentiation. Smad proteins activated by TGF-β form a complex with FoxO proteins to turn on the growth-inhibitory gene p21Cip1 , and BMP-7 induces higher p21 expression than TGF-β1. By interacting with FoxO proteins, therefore, Smad1/4-Shn-2 may also regulate transcription not only of PPARγ2 but also of p21 during adipocyte differentiation (Jin, 2006).

Although more work is required to understand the role of Smad1/4-Shn-2 during adipocyte differentiation, identification of BMP signaling as the key regulatory pathway of adipogenesis in vivo may enable the development of drugs to affect this signaling pathway to suppress obesity and obesity-related diseases (Jin, 2006).