Issue published June 1, 1999

P rolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator–activated receptor α (PPARα) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARα may be involved in the transcriptional response to fasting. To investigate this possibility, PPARα-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARα-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARα-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARα mRNA is induced during fasting in wild-type mice. The data indicate that PPARα plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARα stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

A blation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.J. Clin. Invest. 103:1499–1507 (1999).

P rostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway leading to prostacyclin (PGI₂) production. Patients with severe pulmonary hypertension have a PGIS deficiency of their precapillary vessels, but the importance of this deficiency for lung vascular remodeling remains unclear. We hypothesized that selective pulmonary overexpression of PGIS may prevent the development of pulmonary hypertension. To study this hypothesis, transgenic mice were created with selective pulmonary PGIS overexpression using a construct of the 3.7-kb human surfactant protein-C (SP-C) promoter and the rat PGIS cDNA. Transgenic mice (Tg⁺) and nontransgenic littermates (Tg^–) were subjected to a simulated altitude of 17,000 ft for 5 weeks, and right ventricular systolic pressure (RVSP) was measured. Histology was performed on the lungs. The Tg⁺ mice produced 2-fold more pulmonary 6-keto prostaglandin F_1α (PGF_1α) levels than did Tg^– mice. After exposure to chronic hypobaric hypoxia, Tg⁺ mice have lower RVSP than do Tg^– mice. Histologic examination of the lungs revealed nearly normal arteriolar vessels in the Tg⁺ mice in comparison with vessel wall hypertrophy in the Tg^– mice. These studies demonstrate that Tg⁺ mice were protected from the development of pulmonary hypertension after exposure to chronic hypobaric hypoxia. We conclude that PGIS plays a major role in modifying the pulmonary vascular response to chronic hypoxia. This has important implications for the pathogenesis and treatment of severe pulmonary hypertension.

S tructural changes in the androgen receptor (AR) are one of the causes of defective spermatogenesis. We screened the AR gene of 173 infertile men with impaired spermatogenesis and identified 3 of them, unrelated, who each had a single adenine→guanine transition that changed codon 886 in exon 8 from methionine to valine. This mutation was significantly associated with the severely oligospermic phenotype and was not detected in 400 control AR alleles. Despite the location of this substitution in the ligand-binding domain (LBD) of the AR, neither the genital skin fibroblasts of the subjects nor transfected cell types expressing the mutant receptor had any androgen-binding abnormality. However, the mutant receptor had a consistently (approximately 50%) reduced capacity to transactivate each of 2 different androgen-inducible reporter genes in 3 different cell lines. Deficient transactivation correlated with reduced binding of mutant AR complexes to androgen response elements. Coexpression of AR domain fragments in mammalian and yeast two-hybrid studies suggests that the mutation disrupts interactions of the LBD with another LBD, with the NH₂-terminal transactivation domain, and with the transcriptional intermediary factor TIF2. These data suggest that a functional element centered around M886 has a role, not for ligand binding, but for interdomain and coactivator interactions culminating in the formation of a normal transcription complex.J. Clin. Invest. 103:1517–1525 (1999).

P rotein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4.2–null (4.2^–/–) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2^–/– RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4.2^–/– RBCs are normal. Band 3 and band 3–mediated anion transport are decreased. Protein 4.2^–/– RBCs show altered cation content (increased K⁺/decreased Na⁺)resulting in dehydration. The passive Na⁺ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4.2^–/– RBCs are significantly increased. Protein 4.2^–/– RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2^–/– RBCs compared with controls. The increased passive Na⁺ permeability of 4.2^–/– RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.

P rostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E₂ (PGE₂) receptors — specifically, the EP2 receptor — we show that Ep2^–/– females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2^–/– ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE₂ regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE₂ receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE₂, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum.

O xidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H₂O₂-NO₂^– system of monocytes convert LDL into a form (NO₂-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H₂O₂-generating system, and nitrite (NO₂^–)— a major end-product of NO metabolism—resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO₂-LDL to human monocyte–derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO₂^–, occurred at physiological levels of Cl^–, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO₂-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO₂-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form.

I L-1–stimulated mesenchymal cells model molecular mechanisms of inflammation. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-subunit signaling complex at focal adhesion complexes. Since Rho family GTPases coordinately organize actin cytoskeleton and signaling to regulate cell phenotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved HeLa cells in a Rho-dependent manner and rapidly activated nucleotide exchange on RhoA. Glutathione S-transferase (GST) fusion proteins, containing either the full-length IL-1R cytosolic domain (GST-IL-1Rcd) or the terminal 68 amino acids of IL-1R required for IL-1–dependent signal transduction, specifically coprecipitated both RhoA and Rac-1, but not p21^ras, from Triton-soluble HeLa cell extracts. In whole cells, a small-molecular-weight G protein coimmunoprecipitated by anti–IL-1R antibody was a substrate for C3 transferase, which specifically ADP-ribosylates Rho GTPases. Constitutively activated RhoA, loaded with [γ-³²P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functionally important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd–directed transcriptional activation of the IL-6 gene. Consistent with our previous data demonstrating that IL-1R–associated myelin basic protein (MBP) kinases are necessary for IL-1–directed gene expression, cellular incorporation of C3 transferase inhibited IL-1R–associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically associated with IL-1Rcd and necessary for activation of cytosolic nuclear signaling pathways. These findings suggest that IL-1–stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific architectures that are necessary for persistent cell activation in chronic inflammatory disease.J. Clin. Invest. 103:1561–1570 (1999).

A topic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FcεRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FcεRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FcεRI interaction: Cε3 represents the third constant domain of the IgE heavy chain, and α2 is the membrane-proximal Ig-like module from FcεRIα. Native overlay experiments showed binding of human FcεRIα to recombinant Cε3 and of natural or recombinant human IgE to recombinant α2. Moreover, recombinant Cε3 inhibited binding of natural IgE antibodies to α2, and preincubation of human IgE with α2 inhibited anti-IgE–triggered histamine release from human basophils. Isolated Cε3 and α2 can now be used for the molecular and structural analysis of the IgE-FcεRI interaction, as well as for diagnostic and therapeutic applications.

W e have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform. Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and cholesterol in their diet. However, on all diets tested, the 4/4 mice had approximately twice the amount of cholesterol, apo E, and apo B-48 in their VLDL as did 3/3 mice. The 4/4 VLDL competed with human LDL for binding to the human LDL receptor slightly better than 3/3 VLDL, but the VLDL clearance rate in 4/4 mice was half that in 3/3 mice. On an atherogenic diet, there was a trend toward greater atherosclerotic plaque size in 4/4 mice compared with 3/3 mice. These data, together with our earlier observations in wild-type and human APOE*2-replacement mice, demonstrate a direct and highly significant correlation between VLDL clearance rate and mean atherosclerotic plaque size. Therefore, differences solely in apo E protein structure are sufficient to cause alterations in VLDL residence time and atherosclerosis risk in mice.

K eratins K8 and K18 are the major components of the intermediate-filament cytoskeleton of simple epithelia. Increased levels of these keratins have been correlated with various tumor cell characteristics, including progression to malignancy, invasive behavior, and drug sensitivity, although a role for K8/K18 in tumorigenesis has not yet been demonstrated. To examine the function of these keratins, we generated mice expressing the human K8 (hk8) gene, which leads to a moderate keratin-content increase in their simple epithelia. These mice displayed progressive exocrine pancreas alterations, including dysplasia and loss of acinar architecture, redifferentiation of acinar to ductal cells, inflammation, fibrosis, and substitution of exocrine by adipose tissue, as well as increased cell proliferation and apoptosis. Histological changes were not observed in other simple epithelia, such as the liver. Electron microscopy showed that transgenic acinar cells have keratins organized in abundant filament bundles dispersed throughout the cytoplasm, in contrast to control acinar cells, which have scarce and apically concentrated filaments. The phenotype found was very similar to that reported for transgenic mice expressing a dominant-negative mutant TGF-β type II receptor (TGFβRII mice). We show that these TGFβRII mutant mice also have elevated K8/K18 levels. These results indicate that simple epithelial keratins play a relevant role in the regulation of exocrine pancreas homeostasis and support the idea that disruption of mechanisms that normally regulate keratin expression in vivo could be related to inflammatory and neoplastic pancreatic disorders.

A therosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This “oxidation hypothesis” has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E–deficient mice (apo E^–/–/L-12LO^–/–). In chow-fed 15-week-old apo E^–/–/L-12LO^–/– mice, the extent of lesions in whole-aorta en face preparations (198 ± 60 μm²) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase–expressing controls (apo E^–/–/L-12LO^+/+), which showed areas of lipid deposition (15,700 ± 2,688 μm²) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E–deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E^–/–/L-12LO^–/– mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

A nnexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4⁺ T cells, produced GM-CSF in response to AXII. Furthermore, FACS^® analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4⁺, but not CD8⁺, subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.J. Clin. Invest. 103:1605–1613 (1999).