Function of pref-1 as an inhibitor of adipocyte differentiation.

During conversion of preadipocytes to adipocytes, growth arrest and subsequent activation of adipocyte genes by the transcription factors, C/EBPalpha and PPARgamma, lead to adipogenesis. During differentiation, these cells not only start expressing those genes necessary for adipocyte function, but also undergo changes in morphology to become rounded lipid filled adipocytes. Various factors in cell-cell communication or cell-matrix interaction may govern whether preadipocytes are kept in an undifferentiated state or undergo differentiation. In an attempt to identify molecules that play critical roles in the conversion of preadipocytes to adipocytes, we cloned by differential screening several regulatory molecules, including pref-1. Pref-1 is an inhibitor of adipocyte differentiation and is synthesized as a plasma membrane protein containing 6 EGF-repeats in the extracellular domain. Pref-1 is highly expressed in 3T3-L1 preadipocytes, but is not detectable in mature fat cells. Dexamethasone, a component of standard differentiation agents, inhibits pref-1 transcription and thereby promotes adipogenesis. Downregulation of pref-1 is required for adipose conversion and constitutive expression of pref-1 inhibits adipogenesis. Conversely, decreasing pref-1 levels by antisense pref-1 transfection greatly enhances adipogenesis. The ectodomain of pref-1 is cleaved to generate a biologically active 50kDa soluble form. There are four major forms of membrane pref-1 resulting from alternate splicing. Two of these forms which have a deletion that includes the putative processing site proximal to the membrane do not produce a biologically active soluble form. This indicates that alternate splicing may determine the range of action, juxtacrine or paracrine, of pref-1.

Retinoic acid induces the degradation of the leukemogenic protein encoded by the promyelocytic leukemia gene fused to the retinoic acid receptor alpha gene.

Acute promyelocytic leukemia (APL) cells carry a mutated gene that is the result of a translocation in which the retinoic acid receptor alpha (RAR alpha) gene is fused to the promyelocytic leukemia (PML) gene, coding for a fusion protein, PML/RAR alpha. Its presence is the single event that causes APL in transgenic mice. All-trans-retinoic acid (atRA) induces the proteolytic degradation of PML/RAR alpha by ubiquitination and proteolysis. RAR alpha itself is also degraded by atRA treatment, a process representing a possible feedback mechanism to turn off RAR alpha's stimulation of transcription.

Transcriptional repression of pref-1 by glucocorticoids promotes 3T3-L1 adipocyte differentiation.

Pref-1 is an epidermal growth factor-like domain-containing transmembrane protein that is cleaved to generate a soluble factor. It is abundant in 3T3-L1 preadipocytes but absent in mature adipocytes. Constitutive expression of pref-1 or the addition of its ectodomain inhibits adipogenesis. We find that the pref-1 gene is an early target of dexamethasone, a component of the dexamethasone/methylisobutylxanthine differentiation mixture used routinely for adipoconversion. The time course of the decrease in pref-1 mRNA by dexamethasone reflected the pref-1 mRNA half-life determined by actinomycin D treatment. Nuclear run-on assays showed that dexamethasone attenuates pref-1 transcription. We demonstrate a correlation between pref-1 down-regulation and adipoconversion by varying the time period and concentration of dexamethasone. Increasing the dexamethasone treatment from 2 to 4 days resulted in a time-dependent pref-1 down-regulation and increased differentiation as measured by adipocyte marker mRNAs. The dexamethasone concentration between 1 and 10 nM showed a dose-dependent decrease in pref-1 mRNA and an enhancement of adipogenesis. To test the hypothesis that dexamethasone initiation of adipoconversion may be via down-regulation of pref-1, we lowered endogenous pref-1 mRNA levels by stably transfecting 3T3-L1 preadipocytes with antisense pref-1. At 1 microM, antisense cells had enhanced adipose conversion; a similar degree of differentiation occurred with 2 nM dexamethasone, a concentration that does not support differentiation of control 3T3-L1 cells. We conclude that dexamethasone-mediated repression of pref-1 contributes to the mechanisms whereby glucocorticoids promote adipogenesis.

Transcriptional control of the pref-1 gene in 3T3-L1 adipocyte differentiation. Sequence requirement for differentiation-dependent suppression.

Preadipocyte factor-1 (Pref-1) is a transmembrane epidermal growth factor-like domain-containing protein highly expressed in 3T3-L1 preadipocytes, but is undetectable in mature fat cells; this down-regulation is required for adipocyte differentiation. We show here that pref-1 transcription is markedly suppressed during adipose conversion and results in decreased Pref-1 RNA levels. Using 3T3-L1 cells stably transfected with Pref-1 5'-deletion constructs truncated at -6000, -2100, -1300, -692, -300, -235, -193, -183, -170, -93, and -45 base pairs, we determined that the -183 to -170 region is responsible for the suppression of the pref-1 gene during adipogenesis. This is distinct from the -93 to -45 sequence important for pref-1 promoter activity in preadipocytes. The placement of a 40-base pair -193 to -154 pref-1 sequence containing the putative SAD (suppression in adipocyte differentiation) element upstream of the SV40 promoter decreased promoter activity by 85% upon adipocyte differentiation, compared with 40% observed with the SV40 promoter alone. The SAD element is therefore sufficient for adipocyte differentiation-dependent down-regulation of a heterologous promoter. A DNA-protein complex was observed when the -193 to -174 sequence was used with 3T3-L1 nuclear extracts in gel mobility shift assays. Competition with oligonucleotides harboring base substitution mutations identified a core sequence of -183AAAGA-179 as crucial for DNA-protein complex formation. UV cross-linking predicts that an approximately 63-kDa protein specifically binds the SAD element.

Understanding adipocyte differentiation.

The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR-gamma and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.

Regulation of fat synthesis and adipose differentiation.

Adipocytes have highly specialized function of accumulating fat as stored energy that can be used during periods of food deprivation. The process of fat synthesis and development of adipose tissue are under hormonal and nutritional control. This review first describes transcription of the two critical enzymes involved in fat synthesis, fatty acid synthase and mitochondrial glycerol-3-phosphate acyltransferase, is decreased to an undetectable level during fasting. Food intake, especially a high carbohydrate, fat-free diet, subsequent to fasting causes dramatic increase in transcription of these genes. Insulin secretion is increased during feeding, having a positive effect, whereas cAMP, which mediates the effect of glucagon which increases during fasting, has a negative effect on transcription of these genes. Using adipocytes in culture and in transgenic mice that express liciferase driven by the fatty acid synthase promoter, cis-acting and trans-acting factors that may mediate the transcriptional regulation were examined. Upstream stimulatory factors (USFs) that bind to -65 E-box are required for insulin-mediated transcriptional activation of the fatty acid synthase gene. This review next describes how pref-1 is a novel inhibitor of adipose differentiation and is a plasma membrane protein containing six EGF-repeats in the extracellular domain. Pref-1 is highly expressed in 3T3-L1 preadipocytes, but is not detectable in mature fat cells. Down regulation of pref-1 is required for adipose differentiation, and constitutive expression of pref-1 inhibits adipogenesis. Moreover, the ectodomain of pref-1 is cleaved to generate a biologically active 50 kDa soluble form. There are four major forms of membrane pref-1 resulting from alternate splicing, but two of the forms with a larger deletion do not produce biologically active soluble form, indicating that alternate splicing determines the range of action, juxtacrine or paracrine, of the pref-1.

Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation.

pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state. pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation. Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation. For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa. Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain. We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overriding the adipogenic actions of dexamethasone and methylisobutylxanthine. The inhibitory effects of the pref-1 ectodomain are blocked by preincubation of the protein with pref-1 antibody. That the ectodomain alone is sufficient for inhibition demonstrates that transmembrane pref-1 can be processed to generate an inhibitory soluble form, thereby greatly extending its range of action. Furthermore, we present evidence that alternate splicing is the mechanism that governs the production of transmembrane versus soluble pref-1, thereby determining the mode of action, juxtacrine or paracrine, of the pref-1 protein.

Molecular mechanisms of adipocyte differentiation and inhibitory action of pref-1.

Commitment and differentiation of adipocytes is governed by transcription factors that are under the control of the combinatorial effects of hormonal and cell-cell and cell-matrix interaction. Established preadipocyte cell lines, such as 3T3-L1, 3T3-F442A, and Ob 17, have made it possible to examine the molecular details of the differentiation process. Differentiation is accompanied by dramatic increases in adipocyte genes, including adipocyte fatty acid-binding protein and lipid-metabolizing enzymes. Transcription factors PPAR gamma and C/EBP have been shown to transactivate some of the adipocyte-expressed genes. By integrating hormonal and metabolic cues, these nuclear factors may synergistically function in adipocyte lineage determination and differentiation. Adipocyte differentiation involves drastic cell shape alterations that are accompanied by changes in expression of cytoskeletal and extracellular matrix proteins, including decreases in actin and tubulin levels. Pref-1, an EGF-repeat containing transmembrane protein, is highly expressed in preadipocytes; this expression is totally abolished after differentiation to adipocytes. Pref-1 is inhibitory for adipocyte differentiation and processing of transmembrane pref-1 generates a biologically active soluble from corresponding to the ectodomain. Interaction of the EGF-repeats of pref-1 with an as yet unidentified receptor may mediate the inhibitory effects of pref-1 in adipocyte differentiation, thereby affecting nuclear events accompanying adipogenesis.

Characterization of Pref-1 and its inhibitory role in adipocyte differentiation.

Differentiation of preadipocytes to mature adipocytes is accompanied by marked increases in enzymes of lipid metabolism and cytoskeletal remodelling of fibroblastic preadipocytes to rounded lipid-filled mature adipocytes. Studies to date indicate this process is under the control of specific transcription factors, cell-cell and cell-environment interactions. Preadipocyte factor-1 (pref-1), a member of the EGF-like protein family, was identified during the differential screening of cDNAs expressed in differentiating preadipocytes. Multiple forms of the protein are present in 3T3-L1 preadipocytes with heterogeneity attributable both to alternate splicing and various post-translational modifications. Pref-1 is unique in that its expression is completely abolished during the maturation of preadipocytes to adipocytes. Moreover, blocking down-regulation via constitutive expression of pref-1 in preadipocytes inhibits their differentiation. The expression pattern of pref-1 in embryonic tissues and its presence in tumors indicate that in addition to its function in adipocyte differentiation, pref-1 may have a broader role in differentiation and development.

Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1.

Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C.M., & Sul, H.S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxta-membrane, and predicted transmembrane domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation.

With the aim of identifying novel regulators of adipocyte differentiation, we have cloned and characterized preadipocyte factor 1 (pref-1), a novel member of the epidermal growth factor (EGF)-like family of proteins. Pref-1 is synthesized as a transmembrane protein with six tandem EGF-like repeats. In preadipocytes, multiple discrete forms of pref-1 protein of 45-60 kd are present, owing in part to N-linked glycosylation. While pref-1 mRNA is abundant in preadipocytes, its expression is completely abolished during differentiation of 3T3-L1 preadipocytes to adipocytes. Moreover, constitutive expression of pref-1 in preadipocytes, which in effect blocks its down-regulation, drastically inhibits adipose differentiation. This indicates that pref-1 functions as a negative regulator of adipocyte differentiation, possibly in a manner analogous to EGF-like proteins that govern cell fate decisions in invertebrates.

Regulated expression of the human corticotropin releasing hormone gene by cyclic AMP.

The factors controlling the expression of the hypothalamic neuropeptide, corticotropin releasing hormone (CRH), are poorly understood. We have used a mouse anterior pituitary cell line, AtT-20, permanently transfected with the human CRH gene as a model for studying the regulation of the CRH gene by cyclic AMP. Previously, we demonstrated that in this system the CRH gene is correctly expressed and appropriately negatively regulated by glucocorticoids. Treatment of five CRH-producing cell lines with an activator of adenylate cyclase (forskolin, 0.1-50 microM for 24 h) caused a dose-dependent and specific increase in the amount of CRH mRNA and radioimmunoassay-detectable CRH peptide secreted into the medium. Ribonuclease protection analysis revealed that the CRH gene was transcribed from multiple transcriptional initiation sites located over several hundred nucleotides. Forskolin treatment resulted in a specific increase in the CRH mRNA transcripts initiating from one of these many transcriptional start sites.

A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B.

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins.

Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta.

Corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide involved in the regulation of ACTH secretion, has been detected by RIA in extracts of human placenta. We wished to determine whether this immunoreactive substance is a product of CRH gene expression in the placenta. We have found authentic human CRH (hCRH) mRNA in human placental tissue that is similar in size to hypothalamic CRH mRNA. Furthermore, the transcriptional initiation site for placental hCRH mRNA is identical to that previously predicted for hypothalamic hCRH mRNA, 23-26 nucleotides downstream from a canonical promoter element. Placental hCRH mRNA increases more than 20-fold in the 5 wk preceding parturition, in parallel with a rise in placental hCRH peptide content. These data strongly suggest that the hCRH gene is expressed in the placenta and that this expression changes dramatically during gestation.

Expression and dexamethasone regulation of the human corticotropin-releasing hormone gene in a mouse anterior pituitary cell line.

The factors controlling the expression of corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide involved in the regulation of ACTH secretion, are poorly understood partly because a suitable in vitro model is lacking. To study the regulation of CRH gene expression, an 8-kilobase (kb) DNA fragment containing the entire human CRH gene as well as approximately 6 kb of 5' sequence and 0.8 kb of 3' sequence was isolated from a lambda Charon 4A human genomic library and introduced into a mouse anterior pituitary cell line, AtT-20, by CaPO4 transfection with a neomycin-selectable marker. Approximately 10% of the neomycin-resistant lines stably expressed the CRH gene and secreted radioimmunoassay-detectable CRH into culture media at levels greater than 100 pg/ml. By Southern blot analysis the 8-kb DNA fragment containing the CRH gene had been incorporated intact into the AtT-20 genome. In each CRH-producing strain, but not in the parent AtT-20 cell line, we detected by Northern blot analysis an RNA species that hybridized to two radioactive cRNA probes specific for either the 5' or 3' portion of CRH mRNA, and that co-migrated with placental CRH mRNA. Dexamethasone treatment for 24-96 h caused a specific decrease in CRH mRNA and peptide levels of 40-50% in the five CRH-producing cell lines with half-maximal suppression at approximately 10(-9) M dexamethasone, indicating that CRH gene expression is negatively regulated by glucocorticoids. Thus, we have established an in vitro model suitable for studying in detail those cis- and trans-acting factors which regulate CRH gene expression.