GLUT4 is the major glucose transporter in adipose tissue and skeletal and cardiac muscles. We examined the mechanisms underlying GLUT4 gene expression in 3T3-L1 cells, which express the gene during their differentiation from preadipocytes to adipocytes. In transient transfections, the activity of a mouse GLUT4 promoter extending to -100 bp in the 5'-flanking region did not differ significantly between 3T3-L1 preadipocytes and adipocytes. Promoter activity up to -590 bp in preadipocytes and adipocytes showed a 70% lower and 228% higher activity, respectively, than promoter activity extending to -100 bp. We also examined methylation status of the GLUT4 promoter. Up to -100 bp, there were five CpG sites at -11, -30, -58, -63, and -75 bp. Two CpG sites at -11 and -30 bp were highly methylated in preadipocytes (60 and 92%, respectively) and highly demethylated in adipocytes (28.6 and 25%, respectively). Conversely, three CpG sites at -58, -63, and -75 bp were highly demethylated in both preadipocytes and adipocytes (<12%). In gel mobility-shift assays, a fragment extending from -40 to -1 bp generated a methylation-sensitive band with nuclear extracts from both preadipocytes and adipocytes when the CpG sites were methylated. Southwestern analysis identified a protein of approximately 55 kDa that bound strongly to the methylated probe. Furthermore, methylation of the CpG sites inhibited promoters extending to -50 or -70 bp. These results suggest that in addition to cell type-specific transcription factor, methylation of specific CpG sites and the methylation-sensitive transcription factor contribute to GLUT4 gene regulation during 3T3-L1 differentiation.

The Cyp 2d-9 gene encodes the male-specific steroid 16 alpha-hydroxylase in mouse liver and shares a conserved regulatory element (-100TTCCGGGC-93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92:1302-1306, 1995), the male-preferential demethylation also occurs at CpG/-97 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABP alpha and GABP beta 1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase-GABP fusion proteins bind to the regulatory element only when CpG/-97 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.

We have studied the roles of estrogen receptor-␣ (ER␣) and the Stat5b form of STAT (signal transducers and activators of transcription) in sex-specific expression of Cyp2a4 (steroid 15␣-hydroxylase) and Cyp2d9 (steroid 16␣-hydroxylase) genes using ER␣-deficient mice. ER␣ deficiency resulted in the repression of the female-specific Cyp2a4 and expression of the male-specific Cyp2d9 genes, respectively in females. In ER␣-deficient males, the Cyp2d9 gene continued to be expressed. Nuclear localization of Stat5b occurs in both sexes of ER␣-deficient mice, although it is normally observed in only wildtype males. Nuclear localization of Stat5b correlates with the repression of Cyp2a4 and expression of Cyp2d9, respectively. Because Stat5b was not detectable in liver nuclear extracts prepared from hypophysectomized ER␣-deficient females, the regulation by ER␣ appeared to be mediated through a pituitary hormone (i.e., growth hormone). Thus, ER␣ appears to play a key role in the mechanism that inhibits nuclear localization of Stat5b in female mice, leading to feminization of a ER␣-GHStat5b pathway and Cyp expression. Defaulting to this ER␣-dependent mechanism results in localization of Stat5b to nuclei, which masculinizes the expression of Cyp genes in male mice.Hepatic metabolism of steroids and xenochemicals is sexually dimorphic in rodents and other animals . Sex-specific metabolism is catalyzed by cytochrome P-450s (CYPs) that are expressed either in male or female animals. Certain metabolism by sex-specific CYPs may lead to sex-dependent susceptibility for chemical toxicity and carcinogenicity (Waxman and Chang, 1995a and references therein). Various molecular and/or cellular mechanisms that may regulate transcription of sex-specific CYP genes have been proposed: DNA methylation (Yokomori et al., 1995), hepatocyte nuclear factor 6 (Lahuna et al., 1997) and the Stat5b form of STAT (signal transducers and activators of transcription) (Subramanian et al., 1995;Udy et al., 1997;Teglund et al., 1998). However, defining the regulatory mechanism of sex-specific CYP expression remains to be a major interest in continuous investigations.Hormonally, growth hormone (GH) is known to play a central role in regulating sex-specific CYP genes. Gustafsson and Stenberg (1976) first suggested the existence of a pituitary factor that feminizes steroid metabolism by rat liver microsomes and called it "a feminization factor". Later, this pituitary factor was found to be GH (Kramer and Colby, 1976). In rats, pulsatile GH secretion (in males) is required for activation of the male-specific CYP2C11 gene, whereas continuous GH secretion (in females) is essential for activating the female-specific CYP2C12 gene (Mode et al., 1981). CYP2A4 and CYP2D9 are the well-characterized sex-specific mouse steroid hydroxylases; the former is the female-specific steroid 15␣-hydroxylase, whereas the latter is the male-specific steroid 16␣-hydroxylase Negishi, 1984a,b, 1988). Using GH-deficient Little mice, we previously showed that GH activates and...

Recent data have shown that ferritin, a ubiquitous protein, has a role as a regulator of cellular differentiation. In the present study we have investigated the expression of ferritin mRNAs in cultured C6 cells, a rat glioma cell line, in response to insulin, which has an important role in cellular growth and differentiation. Insulin stimulated steady state levels of both ferritin heavy chain and ferritin light chain mRNAs. An increase in the level of ferritin heavy or light chain mRNA was detected after 2 h of incubation with insulin, and a plateau was reached after 48 h for heavy chain mRNA and after 72 h for light chain mRNA. The responses were dose-dependent and were maximal at 100 nM for both mRNAs. Treatment of cells with actinomycin-D showed that insulin had no effect on the posttranscriptional stability of these mRNAs. Actinomycin-D inhibited insulin-induced accumulation of both mRNAs, suggesting transcriptional stimulation of ferritin genes by insulin. A nuclear run-on assay showed that the insulin-induced increase in ferritin heavy chain mRNA was due to an increase in the rate of gene transcription. We also demonstrated that insulin-like growth factor-I (IGF-I) increased ferritin heavy and light chain mRNA levels in a dose-dependent fashion, and that the maximum effect was obtained at a concentration of 10 nM on both mRNA levels. IGF-I was not only 10-fold more potent, but the absolute level of maximum stimulation was also about 2-fold greater than that for insulin. The combination of insulin (100 nM) and IGF-I (10 nM) showed no additive effect. The results suggested that the ferritin heavy and light chain genes are transcriptionally regulated by insulin and influenced by IGF-I.

Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the malespecific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Sip gene.

During the differentiation of 3T3-L1 preadipocytes to adipocytes, transcriptional activators and repressors regulate the expression of many adipocyte-specific genes [1±4]. In addition, DNA methylation could also play an important role during the process of adipocyte differentiation. We recently showed that methylation of specific CpG sites of the GLUT4 gene and a methylation sensitive transcription factor, contribute to GLUT4 gene regulation during 3T3-L1 differentiation [5]. Similar mechanisms could also regulate the expression of other genes induced during adipocyte differentiation.Leptin, a major hormonal regulator of appetite and fat cell mass, is one of these adipocyte-specific genes [6,7]. It is secreted by adipose tissue in response to a high content of body fat. Thus, mice lacking a functional leptin gene develop hyperphagia, hy- Abstract Aims/hypothesis. The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating the expression of the leptin gene, we examined the methylation status and methylation-sensitive transcription factors during 3T3-L1 differentiation. Methods. DNase I footprinting, electrophoretic mobility-shift assays, and a Southwestern analysis were carried out using nuclear extracts from preadipocytes and adipocytes. Promoter activity was measured by luciferase assays. The CpG methylation pattern was determined.Results. Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt ±161, all of which were highly methylated ( > 92 %) in preadipocytes. Seven of these sites showed a varying degree of demethylation during differentiation, while the site at nt ±54 remained methylated. In electrophoretic mobilityshift assays, DNA fragments from nt ±115 to nt ±70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kM r protein that binds strongly to the methylated probes. Promoter activity was reduced by methylation of the CpG sites up to nt ±115, but not up to nt ±70. Conclusion/interpretation. These results suggest that methylation of specific CpG sites between nt ±115 and nt ±70 and a methylation-sensitive protein could contribute to leptin gene expression during adipocyte differentiation in 3T3-L1 cells. [Diabetologia (2002) 45: 140±148]

The expression of thyrotropin receptor (TSH-R) on various cells derived from bone, including osteoblast-like rat osteosarcoma cells (UMR106 cells), was investigated. TSH receptor mRNA was detected in UMR106 cells by Northern blot analysis. 125I-labeled TSH binding analysis revealed specific high- and low-affinity binding sites (association constants of 5.6 x 10(9) M(-1) and 3.0 x 10(7) M(-1), respectively) on UMR106 cells. Recombinant TSH, but not recombinant human chorionic gonadotropin, increased cyclic adenosine monophosphate (cAMP) production in a concentration-dependent manner in these cells. Furthermore, immunoglobulin Gs from patients with Graves' disease induced cAMP response in UMR106 cells, and the cAMP response index in this cell line correlated with thyroid-stimulating antibody (TSAb) activity detected by Chinese hamster ovary (CHO)-K1 cells transfected with rat TSH-R. We have also demonstrated that recombinant TSH increased cAMP production in human osteoblast-like osteosarcoma (MG63) cells and mouse primary osteoblastic cells. These results suggest that osteoblasts possess functional TSH-R and that abnormal bone metabolism in Graves' disease may be partly explained by the interaction of TSAb with TSH-R in osteoblasts in some patients.

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