Issue published February 1, 2000

S pecialized forms of physiologic cell death lacking certain characteristic morphologic features of apoptosis occur in terminally differentiating tissues, such as in the outer cell layers of epidermis. In these cell layers, NF-κB translocates from the cytoplasm to the nucleus and induces target gene expression. In light of its potent role in regulating apoptotic cell death in other tissues, NF-κB activation in these cells suggests that this transcription factor regulates cell death during terminal differentiation. Here, we show that NF-κB protects normal epithelial cells from apoptosis induced by both TNFα and Fas, whereas NF-κB blockade enhances susceptibility to death via both pathways. Expression of IκBαM under control of keratin promoter in transgenic mice caused a blockade of NF-κB function in the epidermis and provoked premature spontaneous cell death with apoptotic features. In normal tissue, expression of the known NF-κB–regulated antiapoptotic factors, TRAF1, TRAF2, c-IAP1, and c-IAP2, is most pronounced in outer epidermis. In transgenic mice, NF-κB blockade suppressed this expression, whereas NF-κB activation augmented it, consistent with regulation of cell death by these NF-κB effector proteins. These data identify a new role for NF-κB in preventing premature apoptosis in cells committed to undergoing physiologic cell death and indicate that, in stratified epithelium, such cell death normally proceeds via a distinct pathway that is resistant to NF-κB and its antiapoptotic target effector genes.J. Clin. Invest. 105:253–260 (2000).

R eciprocal interactions between vascular endothelial cells and vascular mesenchymal cells are essential for angiogenesis. Here we show that the basic helix-loop-helix transcription factor, dHAND/Hand2, is expressed in the developing vascular mesenchyme and its derivative, vascular smooth muscle cells (VSMCs). Targeted deletion of the dHAND gene in mice revealed severe defects of embryonic and yolk sac vascular development by embryonic day 9.5. Vascular endothelial cells expressed most markers of differentiation. Vascular mesenchymal cells migrated appropriately but failed to make contact with vascular endothelial cells and did not differentiate into VSMCs. In a screen for genes whose expression was dependent upon dHAND (using subtractive hybridization comparing wild-type and dHAND-null hearts), the VEGF₁₆₅ receptor, neuropilin-1, was found to be downregulated in dHAND mutants. These results suggest that dHAND is required for vascular development and regulates angiogenesis, possibly through a VEGF signaling pathway.

I n lipoatrophic diabetes, a lack of fat is associated with insulin resistance and hyperglycemia. This is in striking contrast to the usual association of diabetes with obesity. To understand the underlying mechanisms, we transplanted adipose tissue into A-ZIP/F-1 mice, which have a severe form of lipoatrophic diabetes. Transplantation of wild-type fat reversed the hyperglycemia, dramatically lowered insulin levels, and improved muscle insulin sensitivity, demonstrating that the diabetes in A-ZIP/F-1 mice is caused by the lack of adipose tissue. All aspects of the A-ZIP/F-1 phenotype including hyperphagia, hepatic steatosis, and somatomegaly were either partially or completely reversed. However, the improvement in triglyceride and FFA levels was modest. Donor fat taken from parametrial and subcutaneous sites was equally effective in reversing the phenotype. The beneficial effects of transplantation were dose dependent and required near-physiological amounts of transplanted fat. Transplantation of genetically modified fat into A-ZIP/F-1 mice is a new and powerful technique for studying adipose physiology and the metabolic and endocrine communication between adipose tissue and the rest of the body.

M ultidrug resistance protein 1 (MRP1) is a transporter protein that helps to protect normal cells and tumor cells against the influx of certain xenobiotics. We previously showed that Mrp1 protects against cytotoxic drugs at the testis-blood barrier, the oral epithelium, and the kidney urinary collecting duct tubules. Here, we generated Mrp1/Mdr1a/Mdr1b triple-knockout (TKO) mice, and used them together with Mdr1a/Mdr1b double-knockout (DKO) mice to study the contribution of Mrp1 to the tissue distribution and pharmacokinetics of etoposide. We observed increased toxicity in the TKO mice, which accumulated etoposide in brown adipose tissue, colon, salivary gland, heart, and the female urogenital system. Immunohistochemical staining revealed the presence of Mrp1 in the oviduct, uterus, salivary gland, and choroid plexus (CP) epithelium. To explore the transport function of Mrp1 in the CP epithelium, we used TKO and DKO mice cannulated for cerebrospinal fluid (CSF). We show here that the lack of Mrp1 protein causes etoposide levels to increase about 10-fold in the CSF after intravenous administration of the drug. Our results indicate that Mrp1 helps to limit tissue distribution of certain drugs and contributes to the blood-CSF drug-permeability barrier.

T he thiazolidinedione class of insulin-sensitizing, antidiabetic drugs interacts with peroxisome proliferator–activated receptor γ (PPAR-γ). To gain insight into the role of this nuclear receptor in insulin resistance and diabetes, we conducted metabolic studies in the PPAR-γ gene knockout mouse model. Because homozygous PPAR-γ–null mice die in development, we studied glucose metabolism in mice heterozygous for the mutation (PPAR-γ^+/– mice). We identified no statistically significant differences in body weight, basal glucose, insulin, or FFA levels between the wild-type (WT) and PPAR-γ^+/– groups. Nor was there a difference in glucose excursion between the groups of mice during oral glucose tolerance test, but insulin concentrations of the WT group were greater than those of the PPAR-γ^+/– group, and insulin-induced increase in glucose disposal rate was significantly increased in PPAR-γ^+/– mice. Likewise, the insulin-induced suppression of hepatic glucose production was significantly greater in the PPAR-γ^+/– mice than in the WT mice. Taken together, these results indicate that — counterintuitively — although pharmacological activation of PPAR-γ improves insulin sensitivity, a similar effect is obtained by genetically reducing the expression levels of the receptor.

I nflammation plays an essential role in the initiation and progression of atherosclerosis, but its role in vascular repair after mechanical arterial injury (i.e., percutaneous transluminal coronary angioplasty, PTCA) is unknown. In animal models of vascular injury, leukocytes are recruited as a precursor to intimal thickening. Furthermore, markers of leukocyte activation — in particular, increased expression of the β2-integrin Mac-1 (αMβ2, or CD11b/CD18), which is responsible for firm leukocyte adhesion to platelets and fibrinogen on denuded vessels — predict restenosis after PTCA. To determine whether Mac-1–mediated leukocyte recruitment is causally related to neointimal formation, we subjected mice lacking Mac-1 to a novel form of mechanical carotid artery dilation and complete endothelial denudation. We now report that the selective absence of Mac-1 impairs transplatelet leukocyte migration into the vessel wall, reducing leukocyte accumulation over time. Diminished medial leukocyte accumulation was accompanied by markedly reduced neointimal thickening after vascular injury. These data establish a role for inflammation in neointimal thickening and suggest that leukocyte recruitment to mechanically injured arteries may prevent restenosis.

X enograft recipients produce large amounts of high-affinity anti-Gal IgG in response to Galα1-3Galβ1- 4GlcNAc-R (α-gal) epitopes on the graft. In contrast, ABO-mismatched allograft recipients undergo “accommodation,” a state of very weak immune response to ABO antigens. These differences in anti-carbohydrate immune response were studied in α1,3galactosyltransferase knock-out mice. Pig kidney membranes administered to these mice elicited extensive production of anti-Gal IgG, whereas allogeneic kidney membranes expressing α-gal epitopes elicited only a weak anti-Gal IgM response. Anti-Gal IgG response to xenograft membranes depended on helper T cell activation and was inhibited by anti-CD40L antibody. These T cells were activated by xenopeptides and not by α-gal epitopes. Moreover, allogeneic cell membranes manipulated to express xenoproteins also induced anti-Gal IgG response. Xenoglycoproteins with α-gal epitopes are processed by anti-Gal B cells. Xenopeptides presented by these cells activate a large repertoire of helper T cells required for the differentiation of anti-Gal B cells into cells secreting anti-Gal IgG. Alloglycoproteins with α- gal epitopes have very few immunogenic peptides and fail to activate helper T cells. Similarly, ineffective helper T-cell activation prevents a strong immune response to blood group antigens in ABO-mismatched allograft recipients, thus enabling the development of accommodation.

T he broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1–association with PI 3-kinase defines a key step in insulin resistance.

M utations in the DAX1 gene cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). In affected boys, primary adrenal insufficiency occurs soon after birth or during early childhood; HHG is recognized at the expected time of puberty. In this report, we describe the novel phenotype of a man who presented with apparently isolated adrenal insufficiency at 28 years of age. Examination revealed partial pubertal development and undiagnosed incomplete HHG. Gonadotropin therapy did not improve his marked oligospermia, suggesting a concomitant primary testicular abnormality. Genomic analysis revealed a novel missense mutation, I439S, in DAX1. The mutant DAX-1 protein was studied for its ability to function as a transcriptional repressor of target genes. Consistent with the patient’s mild clinical phenotype, the I439S mutation conferred intermediate levels of repressor activity of DAX-1 when compared with mutations associated with classic AHC. This unique case extends the clinical spectrum of AHC to include delayed-onset primary adrenal insufficiency in adulthood and milder forms of HHG. Furthermore, in accordance with findings in Ahch (Dax1) knockout mice, the clinical features in this patient suggest that DAX-1 function is required for spermatogenesis in humans, independent of its known effects on gonadotropin production.

T he aim of this study was to determine whether colchicine, which has been reported to protect against various hepatotoxic insults, influences the susceptibility of mice to the agonistic anti-Fas antibody, Jo2. All mice that were pretreated with colchicine (2 mg/kg) survived the lethal challenge of intraperitoneal administration of 10 μg of Jo2, whereas all control mice pretreated with γ-lumicolchicine succumbed to the challenge. Twelve micrograms of Jo2 killed less than half of colchicine-pretreated mice and its lethal effects were delayed relative to control mice, which all died within 8 hours. Other microtubule-disrupting agents such as Taxol, vinblastine, and nocodazole also improved the survival of mice treated with the lethal dose of Jo2. Histologic examination showed that colchicine protected against Jo2-induced fulminant liver injury, and TUNEL assay demonstrated that colchicine protected against massive apoptosis of hepatocytes. Hepatocytes isolated from colchicine-pretreated mice exhibited decreased susceptibility to Jo2-induced apoptosis. In addition, colchicine pretreatment reduced surface expression of Fas and decreased Jo2- and TNF-α–induced apoptosis of cultured hepatocytes in the presence of actinomycin D, but did not affect the susceptibility of cultured sinusoidal endothelial cells to Jo2-induced apoptosis. Remarkably, Fas and TNF receptor-1 mRNA and intracellular protein levels increased after colchicine treatment, indicating that colchicine protects against death ligand–induced apoptosis in the liver by decreasing death-receptor targeting to the cell surface.

H eparin-binding epidermal growth factor–like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti– glomerular basement membrane (anti–GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti–HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.

R ecently, we isolated a trypsin-sensitive cholecystokinin-releasing peptide (CCK-RP) from porcine and rat intestinal mucosa. The amino acid sequence of this peptide was determined to be identical to that of the diazepam-binding inhibitor (DBI). To test the role of DBI in pancreatic secretion and responses to feeding, we used pancreaticobiliary and intestinal cannula to divert bile–pancreatic juice from anesthetized rats. Within 2 hours, this treatment caused a 2-fold increase in pancreatic protein output and a >10-fold increase in plasma CCK. Luminal DBI levels increased 4-fold. At 5 hours after diversion of bile–pancreatic juice, each of these measures returned to basal levels. Intraduodenal infusion of peptone evoked a 5-fold increase in the concentration of luminal DBI. In separate studies, we demonstrated that intraduodenal administration of antiserum to a DBI peptide specifically abolished pancreatic secretion and the increase in plasma CCK levels after diversion of bile–pancreatic juice. To demonstrate that DBI mediates the postprandial rise in plasma CCK levels, we showed that intraduodenal administration of 5% peptone induced dramatic increases in pancreatic secretion and plasma CCK, effects that could be blocked by intraduodenal administration of anti-DBI antiserum. Hence, DBI, a trypsin-sensitive CCK-RP secreted from the proximal small bowel, mediates the feedback regulation of pancreatic secretion and the postprandial release of CCK.

A denosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A₃ adenosine receptor (A₃AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A₃AR. Adenosine fails to increase vascular permeability in mast cell–deficient mice, suggesting that this tissue response to adenosine is mast cell–dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcεR1) by antigen, as adenosine is equally effective in mediating these changes in FcεR1 β-chain–deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response.

C -reactive protein (CRP) is involved in host defense, regulation of inflammation, and modulation of autoimmune disease. Although the presence of receptors for CRP on phagocytes has been inferred for years, their identity was determined only recently. FcγRIa, the high-affinity IgG receptor, binds CRP with low affinity, whereas FcγRIIa, the low-affinity IgG receptor, binds CRP with high affinity. Because the single nucleotide polymorphism in FcγRIIA — which encodes histidine or arginine at position 131 — strongly influences IgG2 binding, we determined this polymorphism’s effect on CRP binding. CRP bound with high avidity to monocytes and neutrophils from FcγRIIA R-131 homozygotes, and binding was inhibited by the R-specific mAb 41H16. CRP showed decreased binding to cells from FcγRIIA H-131 homozygotes (which bind IgG2 with high affinity). However, IFN-γ enhanced FcγRI expression by H-131 monocytes and increased CRP binding. FcγRIIa heterozygotes showed intermediate binding. CRP initiated increases in [Ca²⁺]_i in PMN from R-131, but not from H-131 homozygotes. These data provide direct genetic evidence for FcγRIIa as the functional, high-affinity CRP receptor on leukocytes while emphasizing the reciprocal relationship between IgG and CRP binding avidities. This counterbalance may affect the contribution of FcγRIIA alleles to host defense and autoimmunity.

T he CFTR Cl^– channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl^– currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl^– currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II–activated Cl^– currents in these cells.

R etinoid X receptor α–null (RXRα-null) mutants exhibit hypoplasia of their ventricular myocardium and die at the fetal stage. In the present study, we wished to determine whether transgenic re-expression of RXRα in mutant cardiac myocytes could rescue these defects. Two transgenic mouse lines specifically overexpressing an RXRα protein in cardiomyocytes were generated, using the cardiac α-myosin heavy chain (α-MHC) promoter. Breeding the high copy number transgenic line onto an RXRα-null genetic background did not prevent the myocardial hypoplasia and fetal lethality associated with the RXRα^–/– genotype, even though the transgene was expressed in the ventricles as early as 10.5 days post-coitum. These data suggest that the RXRα function involved in myocardial growth may correspond to a non–cell-autonomous requirement forsignal orchestrating the growth and differentiation of myocytes. Interestingly, the adult transgenic mice developed a dilated cardiomyopathy, associated with myofibrillar abnormalities and specific deficiencies in respiratory chain complexes I and II, thus providing an additional model for this genetically complex disease.