David Brownstein

Disruption of Ledgf/Psip1 Results in Perinatal Mortality and Homeotic Skeletal Transformations

ABSTRACT PC4- and SF2-interacting protein 1 (Psip1)—also known as lens epithelium-derived growth factor (Ledgf)—is a chromatin-associated protein that has been implicated in transcriptional regulation, mRNA splicing, and cell survival in vitro, but its biological function in vivo is unknown. We identified an embryonic stem cell clone with disrupted Psip1 in a gene trap screen. The resulting Psip1-βgeo fusion protein retains chromatin-binding activity and the PWWP and AT hook domains of the wild-type protein but is missing the highly conserved C terminus. The majority of mice homozygous for the disrupted Psip1 gene died perinatally, but some survived to adulthood and displayed a range of phenotypic abnormalities, including low fertility, an absence of epididymal fat pads, and a tendency to develop blepharitis. However, contrary to expectations, the lens epithelium was normal. The mutant mice also exhibited motor and/or behavioral defects such as hind limb clenching, reduced grip strength, and reduced locomotor activity. Finally, both Psip1−/− neonates and surviving adults had craniofacial and skeletal abnormalities. They had brachycephaly, small rib cages, and homeotic skeletal transformations with incomplete penetrance. The latter phenotypes suggest a role for Psip1 in the control of Hox expression and may also explain why PSIP1 (LEDGF) is found as a fusion partner with NUP98 in myeloid leukemias.

Enhanced Hippocampal Long-Term Potentiation and Spatial Learning in Aged 11β-Hydroxysteroid Dehydrogenase Type 1 Knock-Out Mice

Glucocorticoids are pivotal in the maintenance of memory and cognitive functions as well as other essential physiological processes including energy metabolism, stress responses, and cell proliferation. Normal aging in both rodents and humans is often characterized by elevated glucocorticoid levels that correlate with hippocampus-dependent memory impairments. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) amplifies local intracellular (“intracrine”) glucocorticoid action; in the brain it is highly expressed in the hippocampus. We investigated whether the impact of 11β-HSD1 deficiency in knock-out mice (congenic on C57BL/6J strain) on cognitive function with aging reflects direct CNS or indirect effects of altered peripheral insulin-glucose metabolism. Spatial learning and memory was enhanced in 12 month “middle-aged” and 24 month “aged” 11β-HSD1−/− mice compared with age-matched congenic controls. These effects were not caused by alterations in other cognitive (working memory in a spontaneous alternation task) or affective domains (anxiety-related behaviors), to changes in plasma corticosterone or glucose levels, or to altered age-related pathologies in 11β-HSD1−/− mice. Young 11β-HSD1−/− mice showed significantly increased newborn cell proliferation in the dentate gyrus, but this was not maintained into aging. Long-term potentiation was significantly enhanced in subfield CA1 of hippocampal slices from aged 11β-HSD1−/− mice. These data suggest that 11β-HSD1 deficiency enhances synaptic potentiation in the aged hippocampus and this may underlie the better maintenance of learning and memory with aging, which occurs in the absence of increased neurogenesis.

Murine gammaherpesvirus-68 infection causes multi-organ fibrosis and alters leukocyte trafficking in interferon-γ receptor knockout mice

Murine gammaherpesvirus-68 (MHV-68) infection in interferon-gamma receptor knockout mice (IFN-gammaR(-)/(-)) results in splenic fibrosis and excessive loss of splenocytes. In our present study we found that MHV-68 infection in IFN-gammaR(-)/(-) mice also resulted in fibrosis and atrophy of the mediastinal lymph nodes, interstitial pulmonary fibrosis and fibrotic changes in the liver. Atrophy and cellular depletion of the spleen in IFN-gammaR(-)/(-) was not the result of increased cell death. The loss of splenocytes in IFN-gammaR(-)/(-) mice, which was most evident on day 23 after infection, correlated with an increase in the number of leukocytes in peripheral blood. At the peak of leukocytosis, on day 23 after infection, peripheral blood cells from infected IFN-gammaR(-)/(-) mice were unable to traffic through the fibrosed spleens of IFN-gammaR(-)/(-) mice but were able to enter the spleens of wild-type mice. This indicates that leukocytosis was in part the result of emigration of cells from the spleen and their subsequent exclusion of re-entry at the height of fibrosis. Significant cytokine and chemokine changes were observed in spleens of IFN-gammaR(-)/(-) mice. IFN-gamma, tumor necrosis factor-alpha (TNF-alpha ), TNF-beta, interleukin-1beta (IL-1beta), transforming growth factor-beta1 (TGF-beta1), lymphotactin, and MIP-1beta were elevated on day 14 after infection whereas chemokines IP-10 and MIG were significantly reduced. These changes suggest a role for dysregulated cytokines and chemokines in severe organ-specific fibrosis with implications for immune-mediated fibrotic disorders.

11β-hydroxysteroid dehydrogenase type 2 deficiency accelerates atherogenesis and causes proinflammatory changes in the endothelium in apoe-/- mice.

Mineralocorticoid receptor (MR) activation is proinflammatory and proatherogenic. Antagonism of MR improves survival in humans with congestive heart failure caused by atherosclerotic disease. In animal models, activation of MR exacerbates atherosclerosis. The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) prevents inappropriate activation of the MR by inactivating glucocorticoids in mineralocorticoid-target tissues. To determine whether glucocorticoid-mediated activation of MR increases atheromatous plaque formation, we generated Apoe(-/-)/11β-HSD2(-/-) double-knockout (E/b2) mice. On chow diet, E/b2 mice developed atherosclerotic lesions by 3 months of age, whereas Apolipoprotein E (Apoe(-/-)) mice remained lesion free. Brachiocephalic plaques in 3-month-old E/b2 mice showed increased macrophage and lipid content and reduced collagen content compared with similar sized brachiocephalic plaques in 6-month-old Apoe(-/-) mice. Crucially, treatment of E/b2 mice with eplerenone, an MR antagonist, reduced plaque development and macrophage infiltration while increasing collagen and smooth muscle cell content without any effect on systolic blood pressure. In contrast, reduction of systolic blood pressure in E/b2 mice using the epithelial sodium channel blocker amiloride produced a less-profound atheroprotective effect. Vascular cell adhesion molecule 1 expression was increased in the endothelium of E/b2 mice compared with Apoe(-/-) mice. Similarly, aldosterone increased vascular cell adhesion molecule 1 expression in mouse aortic endothelial cells, an effect mimicked by corticosterone only in the presence of an 11β-HSD2 inhibitor. Thus, loss of 11β-HSD2 leads to striking atherogenesis associated with activation of MR, stimulating proinflammatory processes in the endothelium of E/b2 mice.

11β-Hydroxysteroid Dehydrogenase Type 1, But Not Type 2, Deficiency Worsens Acute Inflammation and Experimental Arthritis in Mice

Glucocorticoids profoundly influence immune responses, and synthetic glucocorticoids are widely used clinically for their potent antiinflammatory effects. Endogenous glucocorticoid action is modulated by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD). In vivo, 11β-HSD1 catalyzes the reduction of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, thereby increasing intracellular glucocorticoid levels. 11β-HSD2 catalyzes the reverse reaction, inactivating intracellular glucocorticoids. Both enzymes have been postulated to modulate inflammatory responses. In the K/BxN serum transfer model of arthritis, 11β-HSD1-deficient mice showed earlier onset and slower resolution of inflammation than wild-type controls, with greater exostoses in periarticular bone and, uniquely, ganglion cysts, consistent with greater inflammation. In contrast, K/BxN serum arthritis was unaffected by 11β-HSD2 deficiency. In a distinct model of inflammation, thioglycollate-induced sterile peritonitis, 11β-HSD1-deficient mice had more inflammatory cells in the peritoneum, but again 11β-HSD2-deficient mice did not differ from controls. Additionally, compared with control mice, 11β-HSD1-deficient mice showed greater numbers of inflammatory cells in pleural lavages in carrageenan-induced pleurisy with lung pathology consistent with slower resolution. These data suggest that 11β-HSD1 limits acute inflammation. In contrast, 11β-HSD2 plays no role in acute inflammatory responses in mice. Regulation of local 11β-HSD1 expression and/or delivery of substrate may afford a novel approach for antiinflammatory therapy.

A switch in the mechanism of hypertension in the syndrome of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess arises from nonfunctional mutations in 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), an enzyme that inactivates cortisol and confers aldosterone specificity on the mineralocorticoid receptor. Loss of 11betaHSD2 permits glucocorticoids to activate the mineralocorticoid receptor, and the hypertension in the syndrome is presumed to arise from volume expansion secondary to renal sodium retention. An 11betaHSD2 null mouse was generated on an inbred C57BL/6J genetic background, allowing survival to adulthood. 11betaHSD2(-/-) mice had BP approximately 20 mmHg higher on average compared with wild-type mice but were volume contracted, not volume expanded as expected. Initially, impaired sodium excretion associated with increased activity of the epithelial sodium channel was observed. By 80 days of age, however, channel activity was abolished and 11betaHSD2(-/-) mice lost salt. Despite the natriuresis, hypertension remained but was not attributable to intrinsic vascular dysfunction. Instead, urinary catecholamine levels in 11betaHSD2(-/-) mice were double those in wild-type mice, and alpha1-adrenergic receptor blockade rescued the hypertensive phenotype, suggesting that vasoconstriction contributes to the sustained hypertension in this model. In summary, it is proposed that renal sodium retention remains a key event in apparent mineralocorticoid excess but that the accompanying hypertension changes from a renal to a vascular etiology over time.

Translation elongation factor eEF1A2 is essential for post-weaning survival in mice

Translation elongation factor eEF1A, formerly known as EF-1α, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.

Glucocorticoid receptor is required for foetal heart maturation

Glucocorticoids are vital for the structural and functional maturation of foetal organs, yet excessive foetal exposure is detrimental to adult cardiovascular health. To elucidate the role of glucocorticoid signalling in late-gestation cardiovascular maturation, we have generated mice with conditional disruption of glucocorticoid receptor (GR) in cardiomyocytes and vascular smooth muscle cells using smooth muscle protein 22-driven Cre recombinase (SMGRKO mice) and compared them with mice with global deficiency in GR (GR(-/-)). Echocardiography shows impaired heart function in both SMGRKO and GR(-/-) mice at embryonic day (E)17.5, associated with generalized oedema. Cardiac ultrastructure is markedly disrupted in both SMGRKO and GR(-/-) mice at E17.5, with short, disorganized myofibrils and cardiomyocytes that fail to align in the compact myocardium. Failure to induce critical genes involved in contractile function, calcium handling and energy metabolism underpins this common phenotype. However, although hearts of GR(-/-) mice are smaller, with 22% reduced ventricular volume at E17.5, SMGRKO hearts are normally sized. Moreover, while levels of mRNA encoding atrial natriuretic peptide are reduced in E17.5 GR(-/-) hearts, they are normal in foetal SMGRKO hearts. These data demonstrate that structural, functional and biochemical maturation of the foetal heart is dependent on glucocorticoid signalling within cardiomyocytes and vascular smooth muscle, though some aspects of heart maturation (size, ANP expression) are independent of GR at these key sites.

Analysis of a Novel Strain of Murine Gammaherpesvirus Reveals a Genomic Locus Important for Acute Pathogenesis

ABSTRACT Infection of mice by murine gammaherpesvirus 68 (MHV-68) is an excellent small-animal model of gammaherpesvirus pathogenesis in a natural host. We have carried out comparative studies of another herpesvirus, murine herpesvirus 76 (MHV-76), which was isolated at the same time as MHV-68 but from a different murid host, the yellow-necked mouse (Apodemus flavicollis). Molecular analyses revealed that the MHV-76 genome is essentially identical to that of MHV-68, except for deletion of 9,538 bp at the left end of the unique region. MHV-76 is therefore a deletion mutant that lacks four genes unique to MHV-68 (M1, M2,M3, and M4) as well as the eight viral tRNA-like genes. Replication of MHV-76 in cell culture was identical to that of MHV-68. However, following infection of mice, MHV-76 was cleared more rapidly from the lungs. In line with this, there was an increased inflammatory response in lungs with MHV-76. Splenomegaly was also significantly reduced following MHV-76 infection, and much less latent MHV-76 was detected in the spleen. Nevertheless, MHV-76 maintained long-term latency in the lungs and spleen. We utilized a cosmid containing the left end of the MHV-68 genome to reinsert the deleted sequence into MHV-76 by recombination in infected cells, and we isolated a rescuant virus designated MHV-76(cA8+)4 which was ostensibly genetically identical to MHV-68. The growth properties of the rescuant in infected mice were identical to those of MHV-68. These results demonstrate that genetic elements at the left end of the unique region of the MHV-68 genome play vital roles in host evasion and are critical to the development of splenic pathology.

Col4a1 mutation in mice causes defects in vascular function and low blood pressure associated with reduced red blood cell volume

Collagen type IV is the major structural component of the basement membrane and COL4A1 mutations cause adult small vessel disease, familial porencephaly and hereditary angiopathy with nephropathy aneurysm and cramps (HANAC) syndrome. Here, we show that animals with a Col4a1 missense mutation (Col4a1(+/Raw)) display focal detachment of the endothelium from the media and age-dependent defects in vascular function including a reduced response to nor-epinephrine. Age-dependent hypersensitivity to acetylcholine is abolished by inhibition of nitric oxide synthase (NOS) activity, indicating that Col4a1 mutations affect vasorelaxation mediated by endothelium-derived nitric oxide (NO). These defects are associated with a reduction in basal NOS activity and the development of heightened NO sensitivity of the smooth muscle. The vascular function defects are physiologically relevant as they maintain in part the hypotension in mutant animals, which is primarily associated with a reduced red blood cell volume due to a reduction in red blood cell number, rather than defects in kidney function. To understand the molecular mechanism underlying these vascular defects, we examined the deposition of collagen type IV in the basement membrane, and found it to be defective. Interestingly, this mutation also leads to activation of the unfolded protein response. In summary, our results indicate that mutations in COL4A1 result in a complex vascular phenotype encompassing defects in maintenance of vascular tone, endothelial cell function and blood pressure regulation.