Transforming growth interacting factor expression in leiomyoma compared with myometrium.

OBJECTIVE: To investigate the expression of transforming growth interacting factor (TGIF), a Smad transcriptional corepressor, in leiomyoma and matched myometrial tissue samples and the effect of TGIF overexpression in myometrial cells. DESIGN: Experimental study. SETTING: Tertiary university hospital. PATIENT(S): Uterine leiomyoma and myometrial tissues from 16 patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The distribution of TGIF in leiomyoma and myometrial tissues by immunohistochemistry stain, mRNA, and protein expression levels by real-time quantitative polymerase chain-reaction (QPCR) and Western blot. Transcriptional regulation of TGIF in myometrial cells with overexpressed TGIF. RESULT(S): Although TGIF is present in the smooth muscle cells of the leiomyoma and the myometrium, it is not found in the extracellular matrix. The TGIF mRNA and protein expressions were statistically significantly higher in the leiomyoma compared with the matched, unaffected myometrial tissues in both phases of the menstrual cycle. There were no differences in mRNA or protein expression throughout the menstrual cycle. Overexpression of TGIF protein in myometrial cells statistically significantly suppressed up-regulation of plasminogen activator inhibitor (PAI-1) induced by TGF-beta1 treatment. CONCLUSION(S): Expression of TGIF is increased in leiomyoma compared with myometrium. This increase in TGIF expression is not affected by endogenous ovarian hormones. Thus, TGIF is a potential repressor of TGF-beta pathways in myometrial cells.

Is lysyl oxidase-like protein-1, alpha-1 antitrypsin, and neutrophil elastase site specific in pelvic organ prolapse?

INTRODUCTION AND HYPOTHESIS: We investigated whether the expression of alpha-1 antitrypsin (ATT), neutrophil elastase (NE), and lysyl oxidase-like protein 1 (LOXL-1) vary within the vagina in subjects with pelvic organ prolapse (POP). METHODS: Biopsies were obtained from the anterior and posterior vaginal wall of 22 women with POP (> or =stage 2 by POP-Q). The subjects were grouped by the most prominent defect: cystocele, cystocele plus uterine prolapse, and rectocele. Comparative real-time PCR, Western blotting, and NE enzyme activity assay were performed. RESULTS: The ratio of anterior and posterior vaginal wall ATT, NE, and LOXL-1 expression varied between individuals within the same defect group. CONCLUSIONS: ATT, NE, and LOXl-1 expression was variable among different biopsy sites in the vagina. No consistent pattern was present when the subjects were grouped by the most prominent defect. We recommend careful consideration of biopsy sites in future studies on POP to enhance reproducibility of data.

Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation.

Stearoyl-coenzyme A desaturase (SCD) is the rate-limiting enzyme necessary for the biosynthesis of monounsaturated fatty acids. In this study, we investigated the regulation of mouse SCD1 by liver X receptor (LXR) and its role in plasma lipoprotein metabolism upon LXR activation. In vivo, the SCD1 gene remained induced upon LXR activation in the absence of sterol regulatory element-binding protein 1c (SREBP-1c), a known transcriptional regulator of SCD1. Serial deletion and point mutation analyses in reporter gene assays, as well as a gel mobility shift assay, identified an LXR response element in the mouse SCD1 promoter. In addition, SCD1 deficiency prevented the hypertriglyceridemic effect and reduced hepatic triglyceride accumulation associated with LXR activation despite induced hepatic expression of SREBP-1c protein and several SREBP1c and LXR target genes involved in lipoprotein metabolism. Unlike wild-type mice, SCD1-deficient mice failed to elevate the hepatic triglyceride monounsaturated acid (MUFA)/saturated fatty acid (SFA) ratio despite induction of the SCD2 gene. Together, these findings suggest that SCD1 plays a pivotal role in the regulation of hepatic and plasma triglyceride accumulation, possibly by modulating the MUFA-to-SFA ratio. In addition, SCD1 deficiency also increased plasma high-density lipoprotein cholesterol levels induced by LXR activation.

Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis.

Stearoyl-coenzyme A desaturase (SCD) is an endoplasmic reticulum (ER) protein that catalyzes the Delta9-cis desaturation of saturated fatty acids. Mice with targeted disruption in SCD1 (Scd1(-/-)) have significant reduction in the tissue content of triglycerides, suggesting that monounsaturated fatty acids endogenously synthesized by SCD1 are important for triglyceride synthesis. Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is the enzyme that catalyzes the final reaction in the synthesis of triglycerides. The lack of DGAT2, one of the two DGAT isoforms, results in almost a complete loss of tissue triglycerides. We hypothesize that SCD1 participates in triglyceride synthesis by providing a more accessible pool of monounsaturated fatty acids through substrate channeling. In this study, we test whether SCD1 is proximal to DGAT2 by colocalization study with confocal microscopy, coimmunoprecipitation, and fluorescence resonance energy transfer using HeLa cells as the model of study. All of the results suggest that SCD1 and DGAT2 are located very close to each other in the ER, which is a very important criterion for the channeling of substrate. By performing subcellular fractionation using mouse livers, we also show, for the first time, that SCD is present in the mitochondria-associated membrane.

Membrane topology of mouse stearoyl-CoA desaturase 1.

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.

Reduced adiposity and liver steatosis by stearoyl-CoA desaturase deficiency are independent of peroxisome proliferator-activated receptor-alpha.

Stearoyl-CoA desaturase catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids, which are required for normal rates of synthesis of triglycerides, cholesterol esters, and phospholipids. Mice with a targeted disruption of the stearoyl-CoA desaturase 1 (SCD1) isoform are protected against diet and leptin deficiency-induced adiposity, have increased energy expenditure, and have up-regulated expression of hepatic genes encoding enzymes of fatty acid beta-oxidation. Because peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key transcription factor that induces the transcription of fatty acid beta-oxidation and thermogenic genes, we hypothesized that the increased fatty acid oxidation observed in SCD1 deficiency is dependent on activation of the PPARalpha pathway. Here we show that mice nullizygous for SCD1 and PPARalpha are still protected against adiposity, have increased energy expenditure, and maintain high expression of PPARalpha target genes in the liver and brown adipose tissue. The SCD1 deficiency rescued hepatic steatosis of the PPARalpha(-/-) mice. The SCD1 mutation increased the phosphorylation of both AMP-activated protein kinase and acetyl-CoA carboxylase, thereby increasing CPT activity and stimulating the oxidation of liver palmitoyl-CoA in the PPARalpha null mice. The findings indicate that the reduced adiposity, reduced liver steatosis, increased energy expenditure, and increased expression of PPARalpha target genes associated with SCD1 deficiency are independent of activation of the PPARalpha pathway.

Stearoyl-CoA desaturase 1 gene expression is necessary for fructose-mediated induction of lipogenic gene expression by sterol regulatory element-binding protein-1c-dependent and -independent mechanisms.

Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1-/- mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c-/- mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructose-mediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis.

Identification and characterization of murine SCD4, a novel heart-specific stearoyl-CoA desaturase isoform regulated by leptin and dietary factors.

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Thus far, three isoforms of SCD (SCD1, SCD2, and SCD3) have been identified and characterized. Regulation of the SCD1 isoform has been shown to be an important component of the metabolic actions of leptin in liver, but the effects of leptin on SCD isoforms in other tissues have not been investigated. We found that although the mRNA levels of SCD1 and SCD2 were not affected by leptin deficiency in the hearts of ob/ob mice, the SCD activity and levels of monounsaturated fatty acids were increased, implying the existence of another SCD isoform. This observation has led to the cDNA cloning and characterization of a fourth SCD isoform (SCD4) that is expressed exclusively in the heart. SCD4 encodes a 352-amino acid protein that shares 79% sequence identity with the SCD1, SCD2, and SCD3 isoforms. Liver X receptor alpha (LXR alpha) agonists and a high carbohydrate fat-free diet induced SCD4 expression, but unlike SCD1, SCD4 expression was not repressed by dietary polyunsaturated fatty acids. SCD4 mRNA levels were elevated 5-fold in the hearts of leptin-deficient ob/ob mice relative to wild type controls. Treatment of ob/ob mice with leptin decreased mRNA levels of SCD4, whereas levels of SCD1 and SCD2 were not affected. Furthermore, in the hearts of SCD1-deficient mice, SCD4 mRNA levels were induced 3-fold, whereas the levels of SCD2 were not altered. The current studies identify a novel heart-specific SCD isoform that demonstrates tissue-specific regulation by leptin and dietary factors.

Sterol regulatory element-binding proteins (SREBPs) as regulators of lipid metabolism: polyunsaturated fatty acids oppose cholesterol-mediated induction of SREBP-1 maturation.

Cellular cholesterol and fatty acid metabolism in mammals is controlled by a family of transcription factors called sterol regulatory element-binding protein isoforms, three of which (SREBP-1a, 1c, and 2) are well characterized. These proteins, which are synthesized as precursors, are inserted into the endoplasmic reticulum (ER) membrane with both the amino and carboxylic acid domains facing the cytosolic face of the membrane. In sterol-deficient cells, proteolytic cleavage of SREBPs occurs, thereby releasing their N-terminal mature and active forms and enabling them to enter the nucleus, where they bind to the sterol regulatory response element (SRE) and/or E-box sequences and activate genes involved in cholesterol, triglyceride, and fatty acid biosynthesis. Of the three SREBP isoforms, SREBP-1c gene expression is induced by cholesterol and repressed by polyunsaturated fatty acids (PUFA). We have examined the changes in SREBP-1c mRNA and protein levels as well as the mRNA levels of several SREBP-1c target genes when a high-cholesterol diet is combined with diets rich in PUFA of the n-6 series. Our studies show that PUFA oppose the cholesterol-mediated SREBP-1 maturation without affecting the cholesterol-mediated increase of SREBP-1c mRNA and precursor protein. The decrease in SREBP-1 mature protein paralleled the decrease in mRNAs for genes of fatty acid and cholesterol biosynthesis, such as HMG-CoA synthase and fatty acid synthase, but interestingly gene expression of stearoyl-CoA desaturase 1 (SCD1) was instead induced. These studies suggest that the main point of control of PUFA-mediated suppression of lipogenic gene expression is the inhibition of SREBP-1 maturation. The studies also reveal that the induction of SCD1 gene expression by cholesterol occurs through a mechanism independent of SREBP-1 maturation.