Issue published November 1, 2000

C igarette smoke exposure is the major cause of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop significant COPD, and patients with asthma or asthma-like airway hyperresponsiveness or eosinophilia experience accelerated loss of lung function after cigarette smoke exposure. Pulmonary inflammation is a characteristic feature of lungs from patients with COPD. Surprisingly, the mediators of this inflammation and their contributions to the pathogenesis and varied natural history of COPD are not well defined. Here we show that IL-13, a critical cytokine in asthma, causes emphysema with enhanced lung volumes and compliance, mucus metaplasia, and inflammation, when inducibly overexpressed in the adult murine lung. MMP-2, -9, -12, -13, and -14 and cathepsins B, S, L, H, and K were induced by IL-13 in this setting. In addition, treatment with MMP or cysteine proteinase antagonists significantly decreased the emphysema and inflammation, but not the mucus in these animals. These studies demonstrate that IL-13 is a potent stimulator of MMP and cathepsin-based proteolytic pathways in the lung. They also demonstrate that IL-13 causes emphysema via a MMP- and cathepsin-dependent mechanism(s) and highlight common mechanisms that may underlie COPD and asthma.

G rowth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR^–/–), Stat5ab^–/–, and GHR^–/– mice treated with IGF-I. Reduced bone growth in GHR^–/– mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR^–/– mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR^–/– on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab^–/– mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR^–/– mice, indicating that the effects of GH in bone may not involve Stat5 activation.

A polipoprotein J/clusterin (apoJ/clusterin), an intriguing protein with unknown function, is induced in myocarditis and numerous other inflammatory injuries. To test its ability to modify myosin-induced autoimmune myocarditis, we generated apoJ-deficient mice. ApoJ-deficient and wild-type mice exhibited similar initial onset of myocarditis, as evidenced by the induction of two early markers of the T cell–mediated immune response, MHC-II and TNF receptor p55. Furthermore, autoantibodies against the primary antigen cardiac myosin were induced to the same extent. Although the same proportion of challenged animals exhibited some degree of inflammatory infiltrate, inflammation was more severe in apoJ-deficient animals. Inflammatory lesions were more diffuse and extensive in apoJ-deficient mice, particularly in females. In marked contrast to wild-type animals, the development of a strong generalized secondary response against cardiac antigens in apoJ-deficient mice was predictive of severe myocarditis. Wild-type mice with a strong Ab response to secondary antigens appeared to be protected from severe inflammation. After resolution of inflammation, apoJ-deficient, but not wild-type, mice exhibited cardiac function impairment and severe myocardial scarring. These results suggest that apoJ limits progression of autoimmune myocarditis and protects the heart from postinflammatory tissue destruction.

I n collecting duct principal cells, aquaporin 2 (AQP2) is shuttled from intracellular vesicles to the plasma membrane upon vasopressin (VP) stimulation. VP activates adenylyl cyclase, increases intracellular cAMP, activating protein kinase A (PKA) to phosphorylate AQP2 on the COOH-terminal residue, serine 256. Using rat kidney slices and LLC-PK1 cells stably expressing AQP2 (LLC-AQP2 cells), we now show that AQP2 trafficking can be stimulated by cAMP-independent pathways. In these systems, the nitric oxide (NO) donors sodium nitroprusside (SNP) and NONOate and the NO synthase substrate L-arginine mimicked the effect of VP, stimulating relocation of AQP2 from cytoplasmic vesicles to the plasma membrane. Unlike VP, these other agents did not increase intracellular cAMP. However, SNP increased intracellular cGMP, and exogenous cGMP stimulated AQP2-membrane insertion. Atrial natriuretic factor, which signals via cGMP, also stimulated AQP2 translocation. The VP and SNP effects were blocked by the kinase inhibitor H89. SNP did not stimulate membrane insertion of AQP2 in LLC-PK1 cells expressing the phosphorylation-deficient mutant 256SerAla-AQP2, indicating that phosphorylation of Ser256 is required for signaling. Both PKA and cGMP-dependent protein kinase G phosphorylated AQP2 on this COOH-terminal residue in vitro. These results demonstrate a novel, cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion in renal epithelial cells.

T NF-α–induced apoptosis is thought to involve mediators from acidic vesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently been implicated in apoptosis. To determine whether cat B contributes to TNF-α–induced apoptosis, we exposed mouse hepatocytes to the cytokine in vitro and in vivo. Isolated hepatocytes treated with TNF-α in the presence of the transcription inhibitor actinomycin D (AcD) accumulated cat B in their cytosol. Further experiments using cell-free systems indicated that caspase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitochondria. Consistent with these observations, the ability of TNF-α/AcD to induce mitochondrial release of cytochrome c, caspase activation, and apoptosis of isolated hepatocytes was markedly diminished in cells from CatB^–/– mice. Deletion of the CatB gene resulted in diminished liver injury and enhanced survival after treatment in vivo with TNF-α and an adenovirus construct expressing the IκB superrepressor. Collectively, these observations suggest that caspase-mediated release of cat B from lysosomes enhances mitochondrial release of cytochrome c and subsequent caspase activation in TNF-α–treated hepatocytes.

W hile it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle α-actin, smooth muscle myosin heavy chain, or a SM22α promoter–β-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in β-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. β-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22α promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.

M urine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2′-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vβ gene rearrangements and pre–TCR-α expression were normal in ADA-deficient cultures, the production of αβ TCR⁺ thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of β selection. In contrast, the production of γδ TCR⁺ thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor–1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as β selection.

S usceptibility to Alzheimer’s disease (AD) is governed by multiple genetic factors. Remarkably, the LDL receptor–related protein (LRP) and its ligands, apoE and α2M, are all genetically associated with AD. In this study, we provide evidence for the involvement of the LRP pathway in amyloid deposition through sequestration and removal of soluble amyloid β-protein (Aβ). We demonstrate in vitro that LRP mediates the clearance of both Aβ40 and Aβ42 through a bona fide receptor-mediated uptake mechanism. In vivo, reduced LRP expression is associated with LRP genotypes and is correlated with enhanced soluble Aβ levels and amyloid deposition. Although LRP has been proposed to be a clearance pathway for Aβ, this work provides the first in vivo evidence that the LRP pathway may modulate Aβ deposition and AD susceptibility by regulating the removal of soluble Aβ.

P seudohypoparathyroidism type IB (PHPIB) is characterized by renal resistance to parathyroid hormone (PTH) and the absence of other endocrine or physical abnormalities. Familial PHPIB has been mapped to 20q13, near GNAS1, which encodes G_sα, the G protein α-subunit required for receptor-stimulated cAMP generation. However, G_sα function is normal in blood cells from PHPIB patients, ruling out mutations within the G_sα coding region. In mice G_sα is expressed only from the maternal allele in renal proximal tubules (the site of PTH action) but is biallelically expressed in most other tissues. Studies in patients with Albright hereditary osteodystrophy suggest a similar G_sα imprinting pattern in humans. Here we identify a region upstream of the G_sα promoter that is normally methylated on the maternal allele and unmethylated on the paternal allele, but that is unmethylated on both alleles in all 13 PHPIB patients studied. Within this region is an alternative promoter and first exon (exon 1A), generating transcripts that are normally expressed only from the paternal allele, but that are biallelically expressed in PHPIB patients. Therefore, PHPIB is associated with a paternal-specific imprinting pattern of the exon 1A region on both alleles, which may lead to decreased G_sα expression in renal proximal tubules. We propose that loss of exon 1A imprinting is the cause of PHPIB.

W e used expression cloning to isolate cDNAs encoding a microsomal 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase (C₂₇ 3β-HSD) that is expressed predominantly in the liver. The predicted product shares 34% sequence identity with the C₁₉ and C₂₁ 3β-HSD enzymes, which participate in steroid hormone metabolism. When transfected into cultured cells, the cloned C₂₇ 3β-HSD cDNA encodes an enzyme that is active against four 7α-hydroxylated sterols, indicating that a single C₂₇ 3β-HSD enzyme can participate in all known pathways of bile acid synthesis. The expressed enzyme did not metabolize several different C_19/21 steroids as substrates. The levels of hepatic C₂₇ 3β-HSD mRNA in the mouse are not sexually dimorphic and do not change in response to dietary cholesterol or to changes in bile acid pool size. The corresponding human gene on chromosome 16p11.2-12 contains six exons and spans 3 kb of DNA, and we identified a 2-bp deletion in the C27 3β-HSD gene of a patient with neonatal progressive intrahepatic cholestasis. This mutation eliminates the activity of the enzyme in transfected cells. These findings establish the central role of C₂₇ 3β-HSD in the biosynthesis of bile acids and provide molecular tools for the diagnosis of a third type of neonatal progressive intrahepatic cholestasis associated with impaired bile acid synthesis.