Issue published July 15, 2002

P aramyxoviral infections cause most of the acute lower respiratory tract illness in infants and young children and predispose to the development of chronic wheezing, but the relationship between these short- and long-term viral effects are uncertain. Here we show that a single paramyxoviral infection of mice (C57BL6/J strain) not only produces acute bronchiolitis, but also triggers a chronic response with airway hyperreactivity and goblet cell hyperplasia lasting at least a year after complete viral clearance. During the acute response to virus, same-strain ICAM-1–null mice are protected from airway inflammation and hyperreactivity despite similar viral infection rates, but the chronic response proceeds despite ICAM-1 deficiency. Neither response is influenced by IFN-γ deficiency, but the chronic response is at least partially prevented by glucocorticoid treatment. In contrast to viral infection, allergen challenge caused only short-term expression of asthma phenotypes. Thus, paramyxoviruses cause both acute airway inflammation/hyperreactivity and chronic airway remodeling/hyperreactivity phenotypes (the latter by a hit-and-run strategy, since viral effects persist after clearance). These two phenotypes can be segregated by their dependence on the ICAM-1 gene and so depend on distinct controls that appear critical for the development of lifelong airway diseases such as asthma.

T he Polycomb-group (PcG) gene Rae28 is a mammalian homologue of the Drosophila gene polyhomeotic. PcG genes are known to maintain transcription states, once initiated, probably by regulating chromatin structure. Since homozygous Rae28-deficient (Rae28^–/–) mice displayed cardiac anomalies similar to congenital heart diseases in humans, we examined the role of Rae28 in cardiac morphogenesis at the molecular level. In Rae28^–/– embryos, expression of the cardiac selector gene Nkx2.5/Csx (Nkx2.5) was initiated properly but was not sufficiently sustained later in development. This impaired expression of Nkx2.5 in the maintenance phase proved to have a crucial effect on cardiac morphogenesis, as demonstrated by the results of a genetic complementation experiment in which the cardiac anomalies were suppressed by overexpression of human NKX2.5/CSX1 in Rae28^–/– embryos. Ubiquitous expression of exogenous Rae28 likewise restored the impaired Nkx2.5 expression in Rae28^–/– embryos, further supporting the notion that Rae28 sustains Nkx2.5 expression in cardiomyocytes. Thus, our data show that a mammalian PcG gene can play a key role in organogenesis by helping to maintain the expression of a selector gene.

D evelopment of tumor immunotherapies focuses on inducing autoimmune responses against tumor-associated self-antigens primarily encoded by normal, unmutated genes. We hypothesized that such responses could be elicited by T cell homeostatic proliferation in the periphery, involving expansion of T cells recognizing self-MHC/peptide ligands. Herein, we demonstrate that sublethally irradiated lymphopenic mice transfused with autologous or syngeneic T cells showed tumor growth inhibition when challenged with melanoma or colon carcinoma cells. Importantly, the antitumor response depended on homeostatic expansion of a polyclonal T cell population within lymph nodes. This response was effective even for established tumors, was characterized by CD8⁺ T cell–mediated tumor-specific cytotoxicity and IFN-γ production, and was associated with long-term memory. The results indicate that concomitant induction of the physiologic processes of homeostatic T cell proliferation and tumor antigen presentation in lymph nodes triggers a beneficial antitumor autoimmune response.

O ne of the earliest TNF-dependent events to occur during liver regeneration is the activation of the transcription factor NF-κB through TNF receptor type 1. NF-κB activation in the liver can have both antiapoptotic and proliferative effects, but it is unclear which liver cell types, hepatocytes or nonparenchymal cells (NPCs), contribute to these effects. To specifically evaluate the role of hepatocyte NF-κB, we created GLVP/ΔN-IκBα transgenic mice, in which expression of a deletion mutant of IκBα (ΔN-IκBα) was induced in hepatocytes after injection of mifepristone. In control mice, injection of 25 μg/kg TNF caused NF-κB nuclear translocation in virtually all hepatocytes by 30 minutes and no detectable apoptosis, while in mice expressing ΔN-IκBα, NF-κB nuclear translocation was blocked in 45% of hepatocytes, leading to apoptosis 4 hours after TNF injection. In contrast, expression of ΔN-IκBα in hepatocytes during the first several hours after partial hepatectomy did not lead to apoptosis or decreased proliferation. As NF-κB activation was not inhibited in liver NPCs, it is likely that these cells are responsible for mediating the proliferative and antiapoptotic effects of NF-κB during liver regeneration.

K _ATP channels couple the intracellular energy state to membrane excitability and regulate a wide array of biologic activities. K_ATP channels contain a pore-forming inwardly rectifying potassium channel and a sulfonylurea receptor regulatory subunit (SUR1 or SUR2). To clarify the role of K_ATP channels in vascular smooth muscle, we studied Sur2 gene-targeted mice (Sur2^–/–) and found significantly elevated resting blood pressures and sudden death. Using in vivo monitoring, we detected transient, repeated episodes of coronary artery vasospasm in Sur2^–/– mice. Focal narrowings in the coronary arteries were present in Sur2^–/– mice consistent with vascular spasm. We treated Sur2^–/– mice with a calcium channel antagonist and successfully reduced vasospastic episodes.

G raves disease is directly caused by thyroid-stimulating autoantibodies (TSAb’s) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb’s]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb’s, but not for TBAb’s, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

S ex steroid hormone receptors play a central role in all stages of prostate cancer. Here, we tested whether estrogen receptor (ER) signaling contributes to telomerase activation, an early event in prostate tumorigenesis. Following 17β-estradiol (E₂) treatment, both mRNA encoding the catalytic subunit of human telomerase (hTERT) and telomerase activity were promptly induced in human prostate normal epithelial cells, fresh explants from benign prostate hyperplasia, and prostate cancer explants and cell lines. Reporter expression studies and in vivo chromatin immunoprecipitation assays revealed E₂-dependent hTERT promoter induction and showed that both ERα and ERβ bound this sequence. Crucially, addition of the anti-estrogen 4-hydroxytamoxifen caused a differential recruitment in vivo of ERα and ERβ onto the hTERT promoter and inhibited telomerase activity. Treatment with the aromatase inhibitor letrozole, which prevented testosterone-mediated interaction between ER and the hTERT estrogen response element, resulted in a negative regulation of telomerase activity. Thus, intracellular conversion of androgens to estrogens may contribute to the etiopathogenesis of prostate cancer. Given the present evidence for direct control of hTERT gene expression and telomerase activity in the prostate by the ER, we suggest that this transcriptional regulator represents a possible therapeutic target in prostate cancer.

I nappropriate activation of the renin-angiotensin system, which plays a central role in the regulation of blood pressure, electrolyte, and volume homeostasis, may represent a major risk factor for hypertension, heart attack, and stroke. Mounting evidence from clinical studies has demonstrated an inverse relationship between circulating vitamin D levels and the blood pressure and/or plasma renin activity, but the mechanism is not understood. We show here that renin expression and plasma angiotensin II production were increased severalfold in vitamin D receptor–null (VDR-null) mice, leading to hypertension, cardiac hypertrophy, and increased water intake. However, the salt- and volume-sensing mechanisms that control renin synthesis are still intact in the mutant mice. In wild-type mice, inhibition of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] synthesis also led to an increase in renin expression, whereas 1,25(OH)₂D₃ injection led to renin suppression. We found that vitamin D regulation of renin expression was independent of calcium metabolism and that 1,25(OH)₂D₃ markedly suppressed renin transcription by a VDR-mediated mechanism in cell cultures. Hence, 1,25(OH)₂D₃ is a novel negative endocrine regulator of the renin-angiotensin system. Its apparent critical role in electrolytes, volume, and blood pressure homeostasis suggests that vitamin D analogues could help prevent or ameliorate hypertension.

G rowth-arrest specific gene 6 (Gas6) is a vitamin K–dependent growth factor for mesangial and epithelial cells. To investigate whether Gas6 is essential for progressive glomerular injury, we constructed Gas6^–/– mice and examined the role of Gas6 in accelerated nephrotoxic nephritis (NTN), a model of progressive glomerulonephritis. We found less mortality and proteinuria in Gas6^–/– mice than in wild-type mice following injection of nephrotoxic serum. Glomerular cell proliferation, glomerular sclerosis, crescent formation, and deposition of fibrin/fibrinogen in glomeruli were also reduced in Gas6^–/– mice. Furthermore, administering Gas6^–/– mice recombinant wild-type Gas6, but not Gas6 lacking a previously characterized N-terminal γ-carboxyl group, induced massive proteinuria, glomerular cell proliferation, and glomerulosclerosis, comparable to responses seen in wild-type mice. These data indicate that Gas6 induces glomerular cell proliferation in NTN and suggest that this factor contributes to glomerular injury and the progression of chronic nephritis.

G riscelli syndrome (GS) patients and the corresponding mouse model ashen exhibit defects mainly in two types of lysosome-related organelles, melanosomes in melanocytes and lytic granules in CTLs. This disease is caused by loss-of-function mutations in RAB27A, which encodes 1 of the 60 known Rab GTPases, critical regulators of vesicular transport. Here we present evidence that Rab27a function can be compensated by a closely related protein, Rab27b. Rab27b is expressed in platelets and other tissues but not in melanocytes or CTLs. Morphological and functional tests in platelets derived from ashen mice are all within normal limits. Both Rab27a and Rab27b are found associated with the limiting membrane of platelet-dense granules and to a lesser degree with α-granules. Ubiquitous transgenic expression of Rab27a or Rab27b rescues ashen coat color, and melanocytes derived from transgenic mice exhibit widespread peripheral distribution of melanosomes instead of the perinuclear clumping observed in ashen melanocytes. Finally, transient expression in ashen melanocytes of Rab27a or Rab27b, but not other Rab’s, restores peripheral distribution of melanosomes. Our data suggest that Rab27b is functionally redundant with Rab27a and that the pathogenesis of GS is determined by the relative expression of Rab27a and Rab27b in specialized cell types.

T he current dietary treatment of long-chain fatty acid oxidation defects (high carbohydrate with medium-even-chain triglycerides and reduced amounts of long-chain fats) fails, in many cases, to prevent cardiomyopathy, rhabdomyolysis, and muscle weakness. We hypothesized that the apparent defect in energy production results from a depletion of the catalytic intermediates of the citric acid cycle via leakage through cell membranes (cataplerosis). We further hypothesized that replacing dietary medium-even-chain fatty acids (precursors of acetyl-CoA) by medium-odd-chain fatty acids (precursors of acetyl-CoA and anaplerotic propionyl-CoA) would restore energy production and improve cardiac and skeletal muscle function. We fed subjects with long-chain defects a controlled diet in which the fat component was switched from medium-even-chain triglycerides to triheptanoin. In three patients with very-long-chain acyl-CoA dehydrogenase deficiency, this treatment led rapidly to clinical improvement that included the permanent disappearance of chronic cardiomyopathy, rhabdomyolysis, and muscle weakness (for more than 2 years in one child), and of rhabdomyolysis and weakness in the others. There was no evidence of propionyl overload in these patients. The treatment has been well tolerated for up to 26 months and opens new avenues for the management of patients with mitochondrial fat oxidation disorders.

M itogen-activated protein kinase kinase kinase (MEKK1) mediates activation of c-Jun NH₂-terminal kinase (JNK). Although previous studies using cultured cardiac myocytes have suggested that the MEKK1-JNK pathway plays a key role in hypertrophy and apoptosis, its effects in cardiac hypertrophy and apoptosis are not fully understood in adult animals in vivo. We examined the role of the MEKK1-JNK pathway in pressure-overloaded hearts by using mice deficient in MEKK1. We found that transverse aortic banding significantly increased JNK activity in Mekk1^+/+ but not Mekk1^–/– mice, indicating that MEKK1 mediates JNK activation by pressure overload. Nevertheless, pressure overload caused significant levels of cardiac hypertrophy and expression of atrial natriuretic factor in Mekk1^–/– animals, which showed higher mortality and lung/body weight ratio than were seen in controls. Fourteen days after banding, Mekk1^–/– hearts were dilated, and their left ventricular ejection fraction was low. Pressure overload caused elevated levels of apoptosis and inflammatory lesions in these mice and produced a smaller increase in TGF-β and TNF-α expression than occurred in wild-type controls. Thus, MEKK1 appears to be required for pressure overload–induced JNK activation and cytokine upregulation but to be dispensable for pressure overload–induced cardiac hypertrophy. MEKK1 also prevents apoptosis and inflammation, thereby protecting against heart failure and sudden death following cardiac pressure overload.