ER stress and basement membrane defects combine to cause glomerular and tubular renal disease resulting from Col4a1 mutations in mice.

Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies.

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.

Mast cells express 11ß-hydroxysteroid dehydrogenase type 1: a role in restraining mast cell degranulation.

Mast cells are key initiators of allergic, anaphylactic and inflammatory reactions, producing mediators that affect vascular permeability, angiogenesis and fibrosis. Glucocorticoid pharmacotherapy reduces mast cell number, maturation and activation but effects at physiological levels are unknown. Within cells, glucocorticoid concentration is modulated by the 11ß-hydroxysteroid dehydrogenases (11ß-HSDs). Here we show expression and activity of 11ß-HSD1, but not 11ß-HSD2, in mouse mast cells with 11ß-HSD activity only in the keto-reductase direction, regenerating active glucocorticoids (cortisol, corticosterone) from inert substrates (cortisone, 11-dehydrocorticosterone). Mast cells from 11ß-HSD1-deficient mice show ultrastructural evidence of increased activation, including piecemeal degranulation and have a reduced threshold for IgG immune complex-induced mast cell degranulation. Consistent with reduced intracellular glucocorticoid action in mast cells, levels of carboxypeptidase A3 mRNA, a glucocorticoid-inducible mast cell-specific transcript, are lower in peritoneal cells from 11ß-HSD1-deficient than control mice. These findings suggest that 11ß-HSD1-generated glucocorticoids may tonically restrain mast cell degranulation, potentially influencing allergic, anaphylactic and inflammatory responses.

11ß-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 11ß-hydroxysteroid dehydrogenase (11ß-HSD). In vivo, 11ß-HSD1 catalyzes the reduction of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, thereby increasing intracellular glucocorticoid levels. 11ß-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, 11ß-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 11ß-HSD2 deficiency. In a distinct model of inflammation, thioglycollate-induced sterile peritonitis, 11ß-HSD1-deficient mice had more inflammatory cells in the peritoneum, but again 11ß-HSD2-deficient mice did not differ from controls. Additionally, compared with control mice, 11ß-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 11ß-HSD1 limits acute inflammation. In contrast, 11ß-HSD2 plays no role in acute inflammatory responses in mice. Regulation of local 11ß-HSD1 expression and/or delivery of substrate may afford a novel approach for antiinflammatory therapy.

Expression of urocortin peptides in canine myocardium and plasma.

Urocortin (Ucn) peptides are the endogenous ligands for the corticotropin-releasing factor type 2 receptor (CRFR2). They have potentially important roles in cardiovascular physiology in health and disease, and show promise as therapeutics for congestive heart failure. Analysis of canine heart tissue showed mRNA expression of Ucn 1, Ucn 3 and CRFR2 in all heart chambers. Immunohistochemistry also demonstrated Ucns 1 and 3 expression in cardiomyocytes. To assess the potential usefulness of circulating Ucns as markers of heart disease, plasma samples from 45 dogs with cardiac disease and 15 controls were analysed by radioimmunoassay. Both Ucns 1 and 3 were measurable but the presence of cardiac disease did not alter their concentrations. Therefore, whilst Ucns are expressed in canine myocardium (where they may play a role in the endogenous neurohumoral response to cardiac disease or failure) they do not appear to be sensitive biomarkers of cardiac disease in our canine patient population.

11ß-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 11ß-hydroxysteroid dehydrogenase type 2 (11ß-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(-/-)/11ß-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 11ß-HSD2 inhibitor. Thus, loss of 11ß-HSD2 leads to striking atherogenesis associated with activation of MR, stimulating proinflammatory processes in the endothelium of E/b2 mice.

Mice with DNA repair gene Ercc1 deficiency in a neural crest lineage are a model for late-onset Hirschsprung disease.

The Ercc1 gene is essential for nucleotide excision repair and is also important in recombination repair and the repair of interstrand crosslinks. We have previously used a floxed Ercc1 allele with a keratinocyte-specific Cre recombinase transgene to inactivate Ercc1 in the epidermal layer of the skin and so generate a mouse model for UV-induced non-melanoma skin cancer. Now, in an attempt to generate a model for UV-induced melanoma, we have used the floxed Ercc1 allele in combination with a Cre transgene under the control of the tyrosinase gene promoter to produce mice with Ercc1-deficient melanocytes that are hypersensitive to UV irradiation. These animals developed normally, but died when 4-6 months old with severe colonic obstruction. Melanocytes are derived from the neural crest and the tyrosinase promoter is also expressed in additional neural crest-derived lineages, including the progenitors of the parasympathetic nervous system that innervates the gastrointestinal tract and controls gut peristalsis. A functional enteric nervous system developed in floxed Ercc1 mice with the tyrosinase Cre transgene, but was found to have degenerated in the colons of affected mice. We suggest that accumulating unrepaired endogenous DNA damage in the Ercc1-deficient colonic parasympathetic ganglia leads to the degeneration of this network and results in a colonic obstructive disorder that resembles late-onset Hirschsprung disease in man.

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.

Expression of the neuroprotective slow Wallerian degeneration (WldS) gene in non-neuronal tissues.

BACKGROUND: The slow Wallerian Degeneration (Wld(S)) gene specifically protects axonal and synaptic compartments of neurons from a wide variety of degeneration-inducing stimuli, including; traumatic injury, Parkinson's disease, demyelinating neuropathies, some forms of motor neuron disease and global cerebral ischemia. The Wld(S) gene encodes a novel Ube4b-Nmnat1 chimeric protein (Wld(S) protein) that is responsible for conferring the neuroprotective phenotype. How the chimeric Wld(S) protein confers neuroprotection remains controversial, but several studies have shown that expression in neurons in vivo and in vitro modifies key cellular pathways, including; NAD biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress. Whether similar changes are induced in non-neuronal tissue and organs at a basal level in vivo remains to be determined. This may be of particular importance for the development and application of neuroprotective therapeutic strategies based around Wld(S)-mediated pathways designed for use in human patients. RESULTS: We have undertaken a detailed analysis of non-neuronal Wld(S) expression in Wld(S) mice, alongside gravimetric and histological analyses, to examine the influence of Wld(S) expression in non-neuronal tissues. We show that expression of Wld(S) RNA and protein are not restricted to neuronal tissue, but that the relative RNA and protein expression levels rarely correlate in these non-neuronal tissues. We show that Wld(S) mice have normal body weight and growth characteristics as well as gravimetrically and histologically normal organs, regardless of Wld(S) protein levels. Finally, we demonstrate that previously reported Wld(S)-induced changes in cell cycle and cell stress status are neuronal-specific, not recapitulated in non-neuronal tissues at a basal level. CONCLUSIONS: We conclude that expression of Wld(S) protein has no adverse effects on non-neuronal tissue at a basal level in vivo, supporting the possibility of its safe use in future therapeutic strategies targeting axonal and/or synaptic compartments in patients with neurodegenerative disease. Future experiments determining whether Wld(S) protein can modify responses to injury in non-neuronal tissue are now required.

Topical thymidine dinucleotide application protects against UVB-induced skin cancer in mice with DNA repair gene (Ercc1)-deficient skin.

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.

Real-time visualization of human prolactin alternate promoter usage in vivo using a double-transgenic rat model.

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states. Using the dual reporters in combination, dramatic and unexpected changes in PRL expression were observed after inflammatory challenge. Expression of PRL was shown by RT-PCR to be driven by activation of the alternative upstream extrapituitary promoter and flow cytometry analysis pointed at diverse immune cells expressing the reporter gene. These studies demonstrate the effective use of this type of model for molecular physiology and illustrate the potential for providing novel insight into human gene expression using a heterologous system.

Cyp11b1 null mouse, a model of congenital adrenal hyperplasia.

Patients with congenital adrenal hyperplasia arising from mutations of 11beta-hydroxylase, the final enzyme in the glucocorticoid biosynthetic pathway, exhibit glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamo-pituitary-adrenal activity, and excess mineralocorticoid activity caused by the accumulation of deoxycorticosterone. A mouse model, in which exons 3-7 of Cyp11b1 (the gene encoding 11beta-hydroxylase) were replaced with cDNA encoding enhanced cyan fluorescent protein, was generated to investigate the underlying disease mechanisms. Enhanced cyan fluorescent protein was expressed appropriately in the zona fasciculata of the adrenal gland, and targeted knock-out was confirmed by urinary steroid profiles and, immunocytochemically, by the absence of 11beta-hydroxylase. The null mice exhibited glucocorticoid deficiency, mineralocorticoid excess, adrenal hyperplasia, mild hypertension, and hypokalemia. They also displayed glucose intolerance. Because rodents do not synthesize adrenal androgens, changes in reproductive function such as genital virilization of females were not anticipated. However, adult homozygote females were infertile, their ovaries showing an absence of corpora lutea and a central proliferation of disorganized steroidogenic tissue. Null females responded normally to superovulation, suggesting that raised systemic progesterone levels also contribute to infertility problems. The model reveals previously unrecognized phenotypic subtleties of congenital adrenal hyperplasia.

A neurological phenotype in mice with DNA repair gene Ercc1 deficiency.

Transcription-coupled repair of endogenous DNA damage appears crucial for the maintenance of the central and peripheral nervous systems. Ercc1 is essential for nucleotide excision repair and is also involved in recombination repair and the repair of interstrand cross-links. We have investigated the neurological phenotype of Ercc1-deficient mice where the liver dysfunction has been corrected by an Ercc1 transgene controlled by a liver-specific promoter. We observed poor coordination, ataxia and loss of visual acuity, but saw no evidence of the anticipated histopathological neurodegeneration, or of abnormal neuromuscular junctions. Instead we observed uraemic encephalopathy, a brain disease resulting from kidney failure. This diagnosis was supported by histopathological signs of kidney disease, as well as proteinuria. When we examined archival sections from neural-specific Ercc1 knockout mice, which showed the same reduced growth and died at the same age as the liver-corrected Ercc1 knockouts, we found no evidence of kidney pathology or encephalopathy. Thus, while some aspects of the Ercc1-deficient phenotype are indicative of functional neurodegeneration, we obtained no structural evidence for this. The structural changes observed in the brains of liver-corrected Ercc1 knockouts appear to be a secondary consequence of kidney failure arising from Ercc1 deficiency.

A missense mutation in the murine Opa3 gene models human Costeff syndrome.

Opa3 mRNA is expressed in all tissues examined to date, but currently the function of the OPA3 protein is unknown. Intriguingly, various mutations in the OPA3 gene lead to two similar diseases in humans: autosomal dominant inherited optic atrophy and cataract (ADOAC) and a metabolic condition; type 3-methylglutaconic aciduria (MGA). Early onset bilateral optic atrophy is a common characteristic of both disorders; retinal ganglion cells are lost and visual acuity is impaired from an early age. In order to investigate the function of the OPA3 protein, we have generated a novel ENU-induced mutant mouse carrying a missense mutation in the OPA3 gene. The heterozygous mutation in exon 2, causes an amino acid change p.L122P (c.365T>C), which is predicted to alter tertiary protein structure. In the heterozygous state, the mice appear uncompromised however; in the homozygous state mice display some of the features of MGA. Visual function is severely reduced, consistent with significant loss of retinal ganglion cells and degeneration of axons in the optic nerve. In the homozygous optic nerve, there was evidence of increased mitochondrial activity, as demonstrated by the increased presence of mitochondrial marker Cytochrome C Oxidase (COX) histochemistry. Mice homozygous for the opa3(L122P) mutation also display a severe multi-systemic disease characterized by reduced lifespan (majority dying before 4 months), decreased weight, dilated cardiomyopathy, extrapyramidal dysfunction and gross neuro-muscular defects. All of these defects are synonymous with the phenotypic characteristics of Type III MGA found in humans. This model will be of major importance for future studies of the specific function of the OPA3 gene.

Mutationally activated K-ras 4A and 4B both mediate lung carcinogenesis.

To examine the roles of endogenous K-ras 4A and K-ras 4B splice variants in tumorigenesis, murine lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU), which causes a K-ras mutation (G12D) that jointly affects both isoforms. Compared with age-matched K-ras(tmDelta4A/-) mice (where tumours can express mutationally activated K-ras 4B only), tumour number and size were significantly higher in K-ras(+/-) mice (where tumours can also express mutationally activated K-ras 4A), and significantly lower in K-ras(tmDelta4A/tmDelta4A) mice (where tumours can express both wild-type and activated K-ras 4B). MNU induced significantly more, and larger, tumours in wild-type than K-ras(tmDelta4A/tmDelta4A) mice which differ in that only tumours in wild-type mice can express wild-type and activated K-ras 4A. Lung tumours in all genotypes were predominantly papillary adenomas, and tumours from K-ras(+/-) and K-ras(tmDelta4A/-) mice exhibited phospho-Erk1/2 and phospho-Akt staining. Hence (1) mutationally activated K-ras 4B is sufficient to activate the Raf/MEK/ERK(MAPK) and PI3-K/Akt pathways, and initiate lung tumorigenesis, (2) when expressed with activated K-ras 4B, mutationally activated K-ras 4A further promotes lung tumour formation and growth (both in the presence and absence of its wild-type isoform) but does not affect either tumour pathology or progression, and (3) wild-type K-ras 4B, either directly or indirectly, reduces tumour number and size.

Effects on kidney disease, fertility and development in mice inheriting a protein-truncating Denys-Drash syndrome allele (Wt1tmT396).

Denys-Drash syndrome (DDS) is caused by heterozygous mutations of the Wilms' tumour suppressor gene, WT1, characterised by early-onset diffuse mesangial sclerosis often associated with male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that the Wt1tmT396 allele induces DDS kidney disease in mice. In the present study heterozygotes (Wt1tmT396/+) were generated on inbred (129/Ola), crossbred (B6/129) and MF1 second backcross (MF1-N2) backgrounds. Whereas male heterozygotes on each background were fertile, inbred heterozygous females were infertile. Kidney disease (proteinuria and sclerosis) was not congenital and developed significantly earlier in inbred mice, although with variable onset. Disease onset in MF1-N2 stocks occurred later in Wt1tmT396/+ mice than reported previously for Wt1R394W/+ mice, and while no kidney disease has been reported in B6/129 Wt1+/- mice, B6/129 Wt1tmT396/+ mice were affected. Offspring of both male and female B6/129 and MF1-N2 Wt1tmT396/+ mice developed kidney disease, but its incidence was significantly higher in offspring of female heterozygotes. Wt1tmT396/tmT396 embryos exhibited identical developmental abnormalities to those reported for Wt1-/- embryos. The results indicate that the Wt1 (tmT396) allele does not predispose to Wilms' tumourigenesis or male pseudohermaphroditism, its effect on kidney disease and female fertility depends on genetic background, stochastic factors may affect disease onset, and disease transmission is subject to a partial parent-of-origin effect. Since the Wt1tmT396 allele has no detectable intrinsic functional activity in vivo, and kidney disease progression is affected by the type of Wt1 mutation, the data support the view that DDS nephropathy results from a dominant-negative action rather than WT1 haploinsufficiency or gain-of-function.

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.

Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations.

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-betageo 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.

The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice.

Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras(tmDelta4A/tmDelta4A) (exon 4A knock-out) ES cells which express K-ras4B only and K-ras(-/-) (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras(tmDelta4A/tmDelta4A) mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras(tmDelta4A/tmDelta4A) ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras(tmDelta4A/tmDelta4A) and K-ras(-/-) ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras(-/-) cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras(tmDelta4A/tmDelta4A) ES cells were more resistant to etoposide-induced apoptosis than K-ras(-/-) cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.

Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines.

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.