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The reduced bone mineral density (BMD) observed in osteoporosis results, in part, from reduced activity of bone-forming osteoblasts. We examined the effect of peroxisome proliferator-activated receptor (PPAR) activators on MC3T3-E1 preosteoblast maturation. Activators of PPARalpha, delta and gamma induced alkaline phosphatase activity, matrix calcification and the expression of osteoblast genes as determined by reverse transcriptase-polymerase chain reaction. However, at relatively high concentrations of the specific PPARgamma ligands, ciglitazone and troglitazone, maturation was inhibited. PPARalpha, delta and gamma1 were expressed in MC3T3-E1 cells. PPARgamma1 mRNA and protein levels were induced early during osteoblastic maturation. We speculate that endogenous and pharmacological PPAR activators may affect BMD by modulating osteoblastic maturation.

The current studies define the role of three dis- tinct cis -elements in the proximal promoter of the rat farnesyl diphosphate (FPP) synthase gene. The three cis -elements, a sterol regulatory element 3 (SRE-3) flanked by an ATTGG motif (inverted CCAAT box), and a CCAAT box, form a sterol regulatory unit that is necessary and sufficient for sterol-regu- lated expression of FPP synthase promoter-reporter genes. FPP synthase promoter-reporter genes, that contain promot- ers with either wild-type nucleotide sequences or mutations in one or more of the three cis -elements, were transiently trans- fected into CV-1 cells. The activity of the wild-type promoter- reporter gene increased when the cells were incubated in sterol- depleted media or when the cells were co-transfected with a plasmid encoding the mature form of SRE binding protein (SREBP-1a). The results with the mutant promoter-reporter genes demonstrated that all three cis -elements were necessary for normal expression/regulation of the reporter gene by ei- ther sterols or by co-expressed SREBP-1a. Gel mobility shift as- says demonstrated that the synergistic binding of SREBP-1a to SRE-3 was dependent on the binding of recombinant nuclear factor Y (NF-Y) to the DNA, consistent with the in vivo regula- tion studies. —Jackson, S. M., J. Ericsson, R. Mantovani, and P. A. Edwards. Synergistic activation of transcription by nu- clear factor Y and sterol regulatory element binding protein. J. Lipid Res. 1998. 39: 767-776.

The role of the cAMP signaling pathway in vascular calcification was investigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibroblastic morphology and express several osteoblastic differentiation markers. After confluency, they aggregate into cellular condensations, which later mature into nodules where mineralization is localized. Here, we investigated the effects of cAMP on CVC differentiation because it plays a role in both osteoblastic differentiation and vascular disease. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced osteoblast-like "cuboidal" morphology, inhibited proliferation, and enhanced alkaline phosphatase activity, all early markers of osteoblastic differentiation. Isobutylmethylxanthine and cholera toxin had the same effects. Treatment of CVC with pertussis toxin, however, did not induce the morphological change or increase alkaline phosphatase activity, although it inhibited CVC proliferation to a similar extent. cAMP also increased type I procollagen production and gene expression of matrix gamma-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of osteopontin but did not affect the expression of osteocalcin and core binding factor. Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mineralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization during spontaneous CVC differentiation. These results suggest that the cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of CVC.

We demonstrate that the mRNA levels of glycerol-3-phosphate acyltransferase (GPAT), a mitochondrial enzyme catalyzing the initial step in glycerolipid synthesis, are induced during the differentiation of 3T3-L1 preadipocytes to adipocytes and following ectopic expression of rat adipocyte determination and differentiation factor 1 (ADD1), a protein with high homology to the human sterol regulatory element-binding protein-1 (SREBP-1). The increase in GPAT mRNA levels that occurs during differentiation is partially prevented by ectopic expression of a dominant negative form of ADD1. Nucleotide sequences corresponding to the proximal promoter of the murine mitochondrial GPAT gene (Jerkins, A. A., Liu, W. R., Lee, S., and Sul, H. S. (1995) J. Biol. Chem. 270, 1416-1421) bound SREBP-1a and NF-Y in electromobility shift assays. In addition, GPAT promoter-luciferase reporter genes were stimulated by co-expression of SREBP-1a. This increase was attenuated when either a dominant negative form of NF-Y was co-transfected into the cells or when the GPAT promoter contained mutations in the putative binding sites for SREBP-1a or NF-Y. These studies demonstrate that the regulated expression of the mitochondrial GPAT gene requires both NF-Y and ADD1/SREBPs. Thus, SREBPs/ADD1 regulate not only genes involved in cholesterol homeostasis and fatty acid synthesis but also a key enzyme in glycerolipid synthesis.

Sterol-regulated transcription of the farnesyl diphosphate (FPP) synthase gene is dependent on two cis elements in the proximal promoter. These elements, an inverted CCAAT box and sterol regulatory element 3 (SRE-3), bind NF-Y and sterol regulatory element-binding protein 1 (SREBP-1), respectively. We now demonstrate that the binding of recombinant SREBP-1 to its cognate site (SRE-3) within the FPP synthase promoter in vitro is enhanced by binding of NF-Y to the upstream inverted CCAAT box. Using an FPP synthase promoter fragment containing the binding sites for both NF-Y and SREBP-1 in gel mobility shift assays, we demonstrate that the addition of NF-Y increases the binding of SREBP-1 to SRE-3 over 20-fold. In contrast, NF-Y does not stimulate the binding of SREBP-1 to SRE-3 when the inverted CCAAT box is either mutated or 4 base pairs (bp) are inserted between the inverted CCAAT box and SRE-3. Promoter-reporter genes, containing either the wild-type FPP synthase promoter sequence or containing the 4-bp insertion between the inverted CCAAT box and SRE-3, were transiently transfected into cells. The activity of the wild-type promoter-reporter gene increased when the cells were either incubated in sterol-depleted medium or were co-transfected with an expression vector encoding transcriptionally active SREBP-1. This increase in activity was attenuated when the promoter contained the 4-bp insert, consistent with defective binding of SREBP to the promoter in vivo. These studies suggest that the binding of SREBP-1 to SRE-3 in the FPP synthase promoter, and subsequent stimulation of transcription, is dependent on synergistic binding and a functional interaction between SREBP-1 and NF-Y.

In order to define the factors involved in the regulation of farnesyl diphosphate (FPP) synthase, we used sterol regulation-defective (SRD) cell lines that constitutively express either high (SRD-2) or low (SRD-6) levels of transcriptionally active sterol regulatory element binding protein (SREBP). FPP synthase mRNA levels were high in SRD-2 cells and low in SRD-6 cells and were unaffected by the addition or removal of sterols from the media. In contrast, the mRNA levels in parental CHO-7 cells were regulated by sterols. SRD-2, SRD-6, and CHO-7 cells were also transiently transfected with plasmids containing FPP synthase promoter-reporter genes. Reporter gene activity was significantly higher in SRD-2 cells than in either SRD-6 or CHO-7 cells, consistent with a higher rate of transcription of the reporter gene in SRD-2 cells. The high expression of the reporter gene in SRD-2 cells was not observed when the FPP synthase promoter contained a three base pair mutation within an SREBP binding site, termed sterol regulatory element-3 (SRE-3). These observations are consistent with the hypothesis that high levels of transcription of the FPP synthase gene are dependent on the availability of transcriptionally active SREBP. We also demonstrate that the incorporation of radioactive acetate into both cholesterol and fatty acids was enhanced in SRD-2 cells as compared to CHO-7 or SRD-6 cells. Finally, we demonstrate that the concentrations of cholesterol, cholesteryl ester, and triglyceride were all significantly elevated in SRD-2 cells. We conclude that SREBP is involved not only in the regulation of FPP synthase and cholesterogenesis but also in fatty acid and triglyceride synthesis.

The transcription of farnesyl diphosphate (FPP) synthase is regulated up to 30-fold by the sterol status of the cell. Point mutations in a 6-base pair ATTGGC sequence in the promoter disrupt both sterol-dependent transcription in vivo as well as binding of the transcription factor NF-Y in vitro. Co-transfection of cells with NF-YA29, a dominant negative form of NF-Y, and various promoter-reporter genes specifically inhibits the sterol-dependent regulation of FPP synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase. In contrast, NF-YA29 does not affect the regulation of reporter genes under the control of promoters derived from either the HMG-CoA reductase or the low density lipoprotein receptor gene. Transient expression of the 68-kDa transcriptionally active fragment of sterol regulatory element-binding protein in cells stimulates an HMG-CoA synthase-reporter gene over 90-fold. This induction is blocked in cells co-expressing NF-YA29. We hypothesize that NF-Y plays a novel role in sterol-dependent regulation of two key genes in the cholesterol biosynthetic pathway and that this role requires a specific interaction with the sterol regulatory element-binding protein or related transcription factors.

Previous studies identified a 115-base pair (bp) region of the farnesyl diphosphate (FPP) synthase promoter which is involved in the transcriptional regulation of this gene by sterols (Spear, D. H., Kutsunai, S. Y., Correll, C. C., and Edwards, P. A. (1992) J. Biol. Chem. 267, 14462-14469). In the current study we fused a 117-bp fragment, containing this region of interest, upstream of the heterologous minimal promoter of the herpes simplex virus thymidine kinase gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Chinese hamster ovary (CHO) cells were stably transfected with this fusion gene and incubated in the absence or presence of sterols. Analysis of CAT mRNA by primer extension indicated that transcription of the fusion gene was under sterol-mediated control. Thus, when cellular sterols were present, the CAT mRNA levels were reduced 2-4-fold. To further localize the FPP synthase sterol-responsive element(s), additional promoter-reporter gene constructs containing either deletions or mutations were constructed and transfected into CHO or CV-1 cells. These studies localized a 6-bp region (ATTGGC) that is required for both transcriptional induction in the absence of sterols and transcriptional repression in the presence of sterols. Gel shift and footprinting analyses demonstrated that nuclear proteins isolated from CHO cells bound to six distinct regions of the promoter between nucleotides -293 to -47. Taken together, these results further define both the cis-acting elements controlling normal transcription of the FPP synthase gene and identify a novel sequence involved in sterol regulation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels ≈2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR −/− mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

Abstract —Previous studies have demonstrated that the α 1 -adrenergic receptor antagonist doxazosin (Dox) inhibits multiple mitogenic signaling pathways in human vascular smooth muscle cells. This broad antiproliferative activity of Dox occurs through a novel mechanism unrelated to its blocking the α 1 -adrenergic receptor. Flow cytometry demonstrated that Dox prevents mitogen-induced G 1 →S progression of human coronary artery smooth muscle cells (CASMCs) in a dose-dependent manner, with a maximal reduction of S-phase transition by 88±10.5% in 20 ng/mL platelet-derived growth factor and 1 μmol/L insulin (P+I)–stimulated cells ( P P 1 exit by Dox was accompanied by a significant blockade of retinoblastoma protein (Rb) phosphorylation. Hypophosphorylated Rb sequesters the E2F transcription factor, leading to G 1 arrest. Adenoviral overexpression of E2F-1 stimulated quiescent CASMCs to progress through G 1 and enter the S phase. E2F-mediated G 1 exit was not affected by Dox, suggesting that it targets events upstream from Rb hyperphosphorylation. Downregulation of the cyclin-dependent kinase inhibitory protein p27 is important for maximal activation of G 1 cyclin/cyclin-dependent kinase holoenzymes to overcome the cell cycle inhibitory activity of Rb. In Western blot analysis, p27 levels decreased after mitogenic stimulation (after P+I, 43±1.8% of quiescent cells [ P P P P 0 /G 1 phase to the S phase. This G 1 →S blockade likely results from an inhibition of mitogen-induced Rb hyperphosphorylation through prevention of p27 downregulation.

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM-LDL) on osteoblastic differentiation of murine marrow stromal cells, M2-10B4. MM-LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogen-activated protein kinase-dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM-LDL to regulate adipogenesis was also assessed. Treatment of M2-10B4 as well as 3T3-L1 preadipocytes with MM-LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator-activated receptor (PPAR) gamma agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter-reporter construct experiments, MM-LDL may be acting in part through activating PPARalpha. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the "lipid hypothesis of osteoporosis."

Abstract —An early event in acute and chronic inflammation and associated diseases such as atherosclerosis and rheumatoid arthritis is the induced expression of specific adhesion molecules on the surface of endothelial cells (ECs), which subsequently bind leukocytes. Peroxisome proliferator–activated receptors (PPARs), members of the nuclear receptor superfamily of transcription factors, are activated by fatty acid metabolites, peroxisome proliferators, and thiazolidinediones and are now recognized as important mediators in the inflammatory response. Whether PPAR activators influence the inflammatory responses of ECs is unknown. We show that the PPAR activators 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), Wyeth 14643, ciglitazone, and troglitazone, but not BRL 49653, partially inhibit the induced expression of vascular cell adhesion molecule-1 (VCAM-1), as measured by ELISA, and monocyte binding to human aortic endothelial cells (HAECs) activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide. The “natural” PPAR activator 15d-PGJ 2 had the greatest potency and was the only tested molecule capable of partially inhibiting the induced expression of E-selectin and neutrophil-like HL60 cell binding to PMA-activated HAECs. Intracellular adhesion molecule-1 induction by PMA was unaffected by any of the molecules tested. Both PPAR-α and PPAR-γ mRNAs were detected in HAECs by using reverse transcription–polymerase chain reaction and a ribonuclease protection assay; however, we have yet to determine which, if any, of the PPARs are mediating this process. These results suggest that certain PPAR activators may help limit chronic inflammation mediated by VCAM-1 and monocytes without affecting acute inflammation mediated by E-selectin and neutrophil binding.

The isolation and characterization of many of the enzymes involved in the synthesis of cholesterol represented a major tour de force in the 1950s and 1960s (Mead et al., 1986; Porter and Spurgeon, 1981). These early studies led to the realization that the cyclic cholesterol molecule was derived from a polyisoprenoid, which in turn was shown to be composed of repeating five-carbon branched-chain building blocks derived from isopentenyl diphosphate (Rilling, 1985). The condensation of one or more isopentenyl diphosphate molecules with an allylic isoprenoid diphosphate (e.g., dimethylallyl diphosphate, geranyl diphosphate) is catalyzed by a family of related prenyl transferases that differ in their specificity for the allylic substrate (Edwards et al., 1992; Chen et al., 1994). The resulting linear isoprenoids are intermediates in the synthesis of a number of important compounds, including sterols, dolichols, heme a, steroid hormones, 1,25-dihydroxy vitamin D, bile acids, and geranylgeranyl diphosphate (Figure 1). Isoprenoid moieties, such as farnesyl and geranylgeranyl, are also post-translationally attached to certain proteins to produce prenylated proteins (Figure 1). Such modifications affect the localization and functions of the lipidated proteins. Recent studies indicates that isoprenoid alcohols or acids may also functions as novel signaling molecules.

Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.