Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.

Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2-induced oxidative stress in neonatal rat cardiac myocytes.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.

Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained.

Regulation of NF-κB activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((−)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1–1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

Feedback regulation by Atf3 in the endothelin-1-responsive transcriptome of cardiomyocytes: Egr1 is a principal Atf3 target

Endothelin-1 promotes cardiomyocyte hypertrophy by inducing changes in gene expression. Immediate early genes including activating transcription factor 3 (Atf3), Egr1 and Ptgs2 are rapidly and transiently upregulated by endothelin-1 in cardiomyocytes. Atf3 regulates expression of downstream genes and is implicated in negative feedback regulation of other immediate early genes. To identify Atf3-regulated genes, we knocked down Atf3 expression in cardiomyocytes exposed to endothelin-1 and used microarrays to interrogate the transcriptomic effects. Of upregulated mRNAs, expression of 23 (including Egr1, Ptgs2) was enhanced and expression of 25 was inhibited by Atf3 knockdown. Using quantitative PCR, we determined that knockdown of Atf3 had little effect on upregulation of Egr1 mRNA over 30 min, but abolished the subsequent decline, causing sustained Egr1 mRNA expression and enhanced protein expression. This resulted from direct binding of Atf3 to the Egr1 promoter. Mathematical modelling established that Atf3 can suffice to suppress Egr1 expression. Given the widespread co-regulation of Atf3 with Egr1, we suggest that the Atf3-Egr1 negative feedback loop is of general significance. Loss of Atf3 caused abnormal cardiomyocyte growth, presumably resulting from dysregulation of target genes. Our data therefore identify Atf3 as a nexus in cardiomyocyte hypertrophy required to facilitate the full and proper growth response.

Environmental regulation and colonization attributes of the long polar fimbriae (LPF) of Escherichia coli O157:H7

The ability of Escherichia coli O157:H7 to colonize the intestinal epithelia is dependent on the expression of intimin and other adhesins. The chromosome of E. coli O157:H7 carries two loci encoding long polar fimbriae (LPF). These fimbriae mediate adherence to epithelial cells and are associated with colonization of the intestine. In order to increase our knowledge about the conditions controlling their expression and their role in colonization of an animal model, the environmental cues that promote expression of lpf genes and the role of E. coli O157:H7 LPF in intestinal colonization of lambs were investigated. We found that expression of lpf1 was regulated in response to growth phase, osmolarity, and pH; that lpf2 transcription was stimulated during late exponential growth and iron depletion; and that LPF impacts the ability of E. coli O157:H7 to persist in the intestine of infected 6-week-old lambs.

p90 ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to Gq protein-coupled receptor stimuli, endothelin-1 and α1-adrenergic receptor agonists

Extracellular signal-regulated kinases 1/2 (ERK1/2) and their substrates, p90 ribosomal S6 kinases (RSKs), phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes, ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. Here, we investigated the role of RSKs in the transcriptomic responses to Gq protein-coupled receptor agonists, endothelin-1, phenylephrine (generic α1-adrenergic receptor agonist) and A61603 (α1A-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>a61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of 213 RNAs upregulated at 1 h, 51% required RSKs for upregulation whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical to, endothelin-1. As with endothelin-1, PD184352 inhibited upregulation of most phenylephrine-responsive transcripts, but the greater variation in effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α1A-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, upregulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus, RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to Gq protein-coupled receptor stimulation.

RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro

Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate. © 2015 Wiley Periodicals, Inc.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, alpha and beta, exist. The TRalpha and beta isoforms are products of two distinct genes that are further differentially spliced to give TRalpha1 and alpha2, TRbeta1 and beta2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17beta-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERalpha could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERbeta was incapable of inducing the gene in either cell line. ERalpha is therefore not equivalent to ERbeta on this physiological promoter. Indeed, in the neural cell line, ERbeta can inhibit ERalpha-mediated induction from the OTR promoter. While the TRalpha1 isoform inhibited ERalpha-mediated induction in the neural cell line, the TRbeta1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERalpha-mediated induction of the rat OTR gene promoter by the TRalpha1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRalpha1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRalpha-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.

Crosstalk between oestrogen receptors and thyroid hormone receptor isoforms results in differential regulation of the preproenkephalin gene

Nuclear receptors are ligand-activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)alpha-mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand-binding TR isoforms on the ER-mediated induction of the PPE gene in transient transfection assays in CV-1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ER alpha and beta isoforms mediate a four to five-fold induction by oestrogen. Cotransfection of TR alpha 1 along with ER alpha inhibited the ER alpha transactivation of PPE by approximately 50%. However, cotransfection with either TR beta 1 or TR beta 2 expression plasmids produced no effect on the ER alpha or ER beta mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TR alpha 1 DNA-binding mutant could also inhibit ER alpha transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC-1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ER beta is cotransfected, TR alpha 1 stimulated ER beta-mediated transactivation of PPE by approximately eight-fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.

Thyroid hormone can increase estrogen-mediated transcription from a consensus estrogen response element in neuroblastoma cells

Thyroid hormones (T) and estrogens (E) are nuclear receptor ligands with at least two molecular mechanisms of action: (i) relatively slow genomic effects, such as the regulation of transcription by cognate T receptors (TR) and E receptors (ER); and (ii) relatively rapid nongenomic effects, such as kinase activation and calcium release initiated at the membrane by putative membrane receptors. Genomic and nongenomic effects were thought to be disparate and independent. However, in a previous study using a two-pulse paradigm in neuroblastoma cells, we showed that E acting at the membrane could potentiate transcription from an E-driven reporter gene in the nucleus. Because both T and E can have important effects on mood and cognition, it is possible that the two hormones can act synergistically. In this study, we demonstrate that early actions of T via TRalpha1 and TRbeta1 can potentiate E-mediated transcription (genomic effects) from a consensus E response element (ERE)-driven reporter gene in transiently transfected neuroblastoma cells. Such potentiation was reduced by inhibition of mitogen-activated protein kinase. Using phosphomutants of ERalpha, we also show that probable mitogen-activated protein kinase phosphorylation sites on the ERalpha, the serines at position 167 and 118, are important in TRbeta1-mediated potentiation of ERalpha-induced transactivation. We suggest that crosstalk between T and E includes potential interactions through both nuclear and membrane-initiated molecular mechanisms of hormone signaling.

Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells

While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17beta-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Galphaq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERalpha phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERalpha is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBP beta transcriptional activity

Considering that melatonin has been implicated in body weight control, this work investigated whether this effect involves the regulation of adipogenesis. 3T3-L1 preadipocytes were induced to differentiate in the absence or presence of melatonin (10(-3) m). Swiss-3T3 cells ectopically and conditionally (Tet-off system) over-expressing the 34 kDa C/EBP beta isoform (Swiss-LAP cells) were employed as a tool to assess the mechanisms of action at the molecular level. Protein markers of the adipogenic phenotype were analyzed by Western blot. At 36 hr of differentiation of 3T3-L1 preadipocytes, a reduction of PPAR gamma expression was detected followed by a further reduction, at day 4, of perilipin, aP2 and adiponectin protein expression in melatonin-treated cells. Real-time PCR analysis also showed a decrease of PPAR gamma (60%), C/EBP alpha (75%), adiponectin (30%) and aP2 (40%) mRNA expression. Finally, we transfected Swiss LAP cells with a C/EBP alpha gene promoter/reporter construct in which luciferase expression is enhanced in response to C/EBP beta activity. Culture of such transfected cells in the absence of tetracycline led to a 2.5-fold activation of the C/EBP alpha promoter. However, when treated with melatonin, the level of C/EBP alpha promoter activation by C/EBP beta was reduced by 50% (P = 0.05, n = 6). In addition, this inhibitory effect of melatonin was also reflected in the phenotype of the cells, since their capacity to accumulate lipids droplets was reduced as confirmed by the poor staining with Oil Red O. In conclusion, melatonin at a concentration of 10(-3) m works as a negative regulator of adipogenesis acting in part by inhibiting the activity of a critical adipogenic transcription factor, C/EBP beta.