Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.     

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal gene therapy

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2(+/-) mice heterozygous for MnSOD deficiency was compared to that Sod2(+/+) control littermates. Embryo fibroblast cell lines from Sod2(-/-) (neonatal lethal) or Sod2(+/-) mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G(2)/M-phase cell cycle arrest after 5 Gy was observed in Sod2(-/-) cells compared to the Sod2(+/-) or Sod2(+/+) lines. Subclonal Sod2(-/-) or Sod2(+/-) embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2(+/-) mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2(+/+) wild-type littermates. Both Sod2(+/-) and Sod2(+/+) littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2(+/-) knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Mitochondrial localization of superoxide dismutase is required for decreasing radiation-induced cellular damage

We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D0=0.89 +/- 0.01 and 1.08 +/- 0.02 Gy, respectively) compared to parent line 32D cl 3 (D0=1.15 +/- 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D0=2.1 +/- 0.1 and 1.97 +/- 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.     

Regulation of gene expression of epithelial calcium channels in intestine and kidney of mice by 1alpha,25-dihydroxyvitamin D3

In wild-type (VDR(+/+)) mice, ECaC2 expression was confirmed in the intestine and kidney, while ECaC1 expression was exclusively confined to the kidney. Both mRNAs expression of ECaC1 and ECaC2 in the kidney and ECaC2 mRNA expression in the intestine increased time- and dose-dependently in response to 1alpha,25(OH)(2)D(3) injection in VDR(+/+) mice, but not in VDR(-/-) mice. The mRNA levels of ECaC2 in the intestine of VDR(-/-) mice were remarkably reduced when compared to VDR(+/+) mice, while no significant differences were observed in both mRNA levels of ECaC1 and ECaC2 in the kidney between VDR(+/+) mice and VDR(-/-) mice. In the primary renal tubular cells (PRTC) isolated from VDR(+/+) mice, both ECaCs mRNA expression increased in response to 1alpha,25(OH)(2)D(3) treatment, but not in the PRTC of VDR(-/-) mice. PTH increased both ECaCs mRNA expression in the PRTC of VDR(+/+) mice. These results suggest that 1alpha,25(OH)(2)D(3) directly modulates the gene expression of ECaC1 and ECaC2 together with PTH in the kidney of mice. 1alpha,25(OH)(2)D(3) also modulates the gene expression of ECaC2 in the intestine of mice, however, further studies are needed to elucidate the direct action of 1alpha,25(OH)(2)D(3) on the expression of ECaC2 in the intestine.     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Radiation-induced alterations in rat mesangial cell Tgfb1 and Tgfb3 gene expression are not associated with altered secretion of active Tgfb isoforms

Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P 相似文献   

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

Angelica sinensis down-regulates hydroxyproline and Tgfb1 and provides protection in mice with radiation-induced pulmonary fibrosis

Pulmonary fibrosis is a common delayed side effect of radiation therapy, and it has a poor prognosis. Tgfb1 is a potent chemoattractant for fibroblasts and stimulates the production of collagen, the protein that contains hydroxyproline. Since collagen is by far the most abundant protein in the lung, comprising 60-70% of the tissue mass, analysis of the hydroxyproline content in lung tissues provides a reliable quantitative index for pulmonary fibrosis. Thus hydroxyproline and Tgfb1 may be involved in the development of fibrosis. In this study, we investigated radiation-induced pulmonary fibrosis in a mouse model. C57BL/6 mice were assigned into four groups: no treatment, treated with Angelica sinensis treated only, X-irradiated only (a single fraction of 12 Gy to the thorax), and Angelica sinensis treatment plus radiation. We assayed expression of hydroxyproline and the mRNA and protein of Tgfb1 in the four groups. We found that Angelica sinensis down-regulated the production of Tgfb1 and hydroxyproline in mice with radiation-induced pulmonary fibrosis. This study has demonstrated for the first time that Angelica sinensis inhibits the progress of radiation-induced pulmonary fibrosis, possibly by down-regulating the expression of the proinflammatory cytokine Tgfb1. These data suggest that Angelica sinensis may be useful in preventing and/or treating radiation-induced pulmonary fibrosis in the clinic.     

Pirfenidone modulates macrophage polarization and ameliorates radiation-induced lung fibrosis by inhibiting the TGF-β1/Smad3 pathway

Radiation-induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation-induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)-approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X-ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor-β1 (TGF-β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation-induced expression of TGF-β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL-4–induced M2 macrophage polarization and IL-13–induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase-1 (ARG-1), chitinase 3-like 3 (YM-1) and TGF-β1. Notably, the PFD-induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa-B (NF-κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation-induced chemokine secretion in MLE-12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF-β1 from M2 macrophages by attenuating the activation of TGF-β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF-β1/Smad3.     

TP53 and TP53-related genes associated with protection from apoptosis in the radioadaptive response

We investigated the effect of administering priming low-dose radiation prior to high-dose radiation on the level of apoptosis and on the expression of TP53 and TP53-related genes in mouse splenocytes. The percentage of apoptotic cells was significantly lower in TP53(+/+) mice receiving priming radiation 2 to 168 h before the high-dose irradiation, compared to TP53(+/+) mice exposed to 2 Gy alone. In contrast, TP53(+/-) mice exhibited a reduced level of apoptosis only when priming was performed for 2 or 4 h prior to the high-dose irradiation. In TP53(+/+) mice, primed mice had higher TP53 expression than mice exposed to 2 Gy. Phospho-TP53 (ser15/18) expression was the highest in mice exposed to 2 Gy and intermediate in primed mice. Expression of p21 (CDKN1A) was higher in primed mice compared with mice exposed to 2 Gy. MDM2 expression remained at a high level in all mice receiving 2 Gy. Elevated phospho-ATM expression was observed only in mice exposed to 2 Gy. We conclude that TP53 plays a critical role in the radioadaptive response and that TP53 and TP53-related genes might protect cells from apoptosis through activation of the intracellular repair system.     

Cardiac hypertrophy in anion exchanger 1-null mutant mice with severe hemolytic anemia

Anion exchanger 1 (AE1; SLC4A1), the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes, is also expressed in heart. The aim of this study was to assess the role of AE1 in heart function through study of AE1-null (AE1(-/-)) mice, which manifest severe hemolytic anemia resulting from erythrocyte fragility. Heart weight-to-body weight ratios were significantly higher in the AE1(-/-) mice than in wild-type (AE1(+/+)) littermates at both 1-3 days postnatal (3.01 +/- 0.38 vs. 1.45 +/- 0.04) and at 7 days postnatal (9.45 +/- 0.53 vs. 4.13 +/- 0.41), indicating that loss of AE1 led to cardiac hypertrophy. Heterozygous (AE1(+/-)) mice had no signs of cardiac hypertrophy. Morphology of the adult AE1(-/-) mutant heart revealed an increased left ventricular mass, accompanied by increased collagen deposition and fibrosis. M-mode echocardiography revealed dysfunction of the AE1(-/-) hearts, including dilated left ventricle end diastole and systole and expanded left ventricular mass compared with AE1(+/+) hearts. Expression of intracellular pH-regulatory mechanisms in the hypertrophic myocardium of neonate AE1(-/-) mutant mice was indistinguishable from AE1(+/-) and AE1(+/+) mice, as assessed by quantitative real-time RT-PCR. Confocal immunofluorescence revealed that, in normal mouse myocardium, AE1 is sarcolemmal, whereas AE3 and slc26a6 are found both at the sarcolemma and in internal membranes (T tubules and sarcoplasmic reticulum). These results indicate that AE1(-/-) mice, which suffer from severe hemolytic anemia and spherocytosis, display cardiac hypertrophy and impaired cardiac function, reminiscent of findings in patients with hereditary abnormalities of red blood cells. No essential role for AE1 in heart function was found.     

The [Ru(Hedta)NO](0.1-) system: structure, chemical reactivity and biological assays

The [Ru(II)(Hedta)NO(+)] complex is a diamagnetic species crystallizing in a distorted octahedral geometry, with the Ru-N(O) length 1.756(4) A and the RuNO angle 172.3(4) degrees . The complex contains one protonated carboxylate (pK(a)=2.7+/-0.1). The [Ru(II)(Hedta)NO(+)] complex undergoes a nitrosyl-centered one-electron reduction (chemical or electrochemical), with E(NO+/NO)=-0.31 V vs SCE (I=0.2 M, pH 1), yielding [Ru(II)(Hedta)NO](-), which aquates slowly: k(-NO)=2.1+/-0.4x10(-3) s(-1) (pH 1.0, I=0.2 M, CF(3)COOH/NaCF(3)COO, 25 degrees C). At pHs>12, the predominant species, [Ru(II)(edta)NO](-), reacts according to [Ru(II)(edta)NO](-)+2OH(-)-->[Ru(II)(edta)NO(2)](3-), with K(eq)=1.0+/-0.4 x 10(3) M(-2) (I=1.0 M, NaCl; T=25.0+/-0.1 degrees C). The rate-law is first order in each of the reactants for most reaction conditions, with k(OH(-))=4.35+/-0.02 M(-1)s(-1) (25.0 degrees C), assignable mechanistically to the elementary step comprising the attack of one OH(-) on [Ru(II)(edta)NO](-), with subsequent fast deprotonation of the [Ru(II)(edta)NO(2)H](2-) intermediate. The activation parameters were DeltaH(#)=60+/-1 kJ/mol, DeltaS(#)=-31+/-3 J/Kmol, consistent with a nucleophilic addition process between likely charged ions. In the toxicity up-and-down tests performed with Swiss mice, no death was observed in all the doses administered (3-9.08 x 10(-5) mol/kg). The biodistribution tests performed with Wistar male rats showed metal in the liver, kidney, urine and plasma. Eight hours after the injection no metal was detected in the samples. The vasodilator effect of [Ru(II)(edta)NO](-) was studied in aortic rings without endothelium, and was compared with sodium nitroprusside (SNP). The times of maximal effects of [Ru(II)(edta)NO](-) and SNP were 2 h and 12 min, respectively, suggesting that [Ru(II)(edta)NO](-) releases NO slowly to the medium in comparison with SNP.     

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-β signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-β/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-β/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.     

Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.     

Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/-0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.     

Genetic removal of Smad3 from inhibin-null mice attenuates tumor progression by uncoupling extracellular mitogenic signals from the cell cycle machinery

Inhibin and activin are members of the TGFbeta family that perform mutually antagonistic signaling roles in the anterior pituitary, gonads, and adrenal gland. Unopposed activin signaling in inhibin-null (Inha-/-) mice causes the formation of granulosa cell tumors in the gonads and adrenal cortex, which depend upon FSH for efficient growth and progression. In this study, we demonstrate that Smad3, a key effector of activin signaling, is expressed at high levels and is constitutively activated in tumors from these mice. Removal of Smad3 from Inha-/- mice by a genetic cross to Smad3-null (Madh3-/-) mice leads to a significant decrease in cyclinD2 expression and a significant attenuation of tumor progression in the gonads and adrenal. The decrease in cyclinD2 levels in compound knockout mice is related to a reduction in mitogenic signaling through the phosphoinositide-3-kinase (PI3-kinase)/Akt pathway, which is required for normal cell cycle progression in tumor cells. Loss of PI3-kinase/Akt signaling cannot be attributed to alterations in IGF expression, suggesting instead that signaling through the FSH receptor is attenuated. Gene expression profiling in the ovaries of Madh3-/- and Inha-/-:Madh3-/- compound knockout mice supports this hypothesis and further suggests that Smad3 is specifically required for FSH to activate PI3-kinase/Akt, but not protein kinase A. Together these observations imply that activin/Smad3 signaling is necessary for efficient signaling by FSH in Inha-/- tumor cells and that interruption of this pathway uncouples FSH from its intracellular mitogenic effectors.     

Genetic deficiency of Smad3 protects against murine ischemic acute kidney injury

TGF-β1 contributes to chronic kidney disease, at least in part, via Smad3. TGF-β1 is induced in the kidney following acute ischemia, and there is increasing evidence that TGF-β1 may protect against acute kidney injury. As there is a paucity of information regarding the functional significance of Smad3 in acute kidney injury, the present study explored this issue in a murine model of ischemic acute kidney injury in Smad3(+/+) and Smad3(-/-) mice. We demonstrate that, at 24 h after ischemia, Smad3 is significantly induced in Smad3(+/+) mice, whereas Smad3(-/-) mice fail to express this protein in the kidney in either the sham or postischemic groups. Compared with Smad3(+/+) mice, and 24 h following ischemia, Smad3(-/-) mice exhibited greater preservation of renal function as measured by blood urea nitrogen (BUN) and serum creatinine; less histological injury assessed by both semiquantitative and qualitative analyses; markedly suppressed renal expression of IL-6 and endothelin-1 mRNA (but comparable expression of MCP-1, TNF-α, and heme oxygenase-1 mRNA); and no increase in plasma IL-6 levels, the latter increasing approximately sixfold in postischemic Smad3(+/+) mice. We conclude that genetic deficiency of Smad3 confers structural and functional protection against acute ischemic injury to the kidney. We speculate that these effects may be mediated through suppression of IL-6 production. Finally, we suggest that upregulation of Smad3 after an ischemic insult may contribute to the increased risk for chronic kidney disease that occurs after acute renal ischemia.     

Renal function of gene-targeted mice lacking both SGK1 and SGK3

Serum- and glucocorticoid-inducible kinase (SGK) 1 and SGK3 share the ability to upregulate several ion channels, including the epithelial Na(+) channel. Whereas SGK1 is under genomic control of mineralocorticoids and glucocorticoids, SGK3 is constitutively expressed. The SKG1-knockout (sgk1(-/-)) mouse is seemingly normal when it is fed a standard diet, but its ability to retain NaCl is impaired when it is fed a salt-deficient diet. In the SGK3-knockout (sgk3(-/-)) mouse fed standard and salt-deficient diets, hair growth is strikingly delayed but NaCl excretion is normal. Thus the possibility was considered that SGK1 and SGK3 could mutually replace each other, thus preventing severe NaCl loss in sgk1(-/-) and sgk3(-/-) mice. We crossed SGK1- and SGK3-knockout mice and compared renal electrolyte excretion of the double mutants (sgk1(-/-)/sgk3(-/-)) with that of their wild-type littermates (sgk1(+/+)/sgk3(+/+)). Similar to sgk3(-/-) mice, the sgk1(-/-)/sgk3(-/-) mice display delayed hair growth. Blood pressure was slightly, but significantly (P < 0.03), lower in sgk1(-/-)/sgk3(-/-) (102 +/- 4 mmHg) than in sgk1(+/+)/sgk3(+/+) (114 +/- 3 mmHg) mice, a difference that was maintained in mice fed low- and high-salt diets. Plasma aldosterone concentrations were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) than in sgk1(+/+)sgk3(+/+) mice fed control (511 +/- 143 vs. 143 +/- 32 pg/ml) and low-salt (1,325 +/- 199 vs. 362 +/- 145 pg/ml) diets. During salt depletion, absolute and fractional excretions of Na(+) were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) (1.2 +/- 0.2 micromol/24 h g body wt, 0.12 +/- 0.03%) than in sgk1(+/+)/sgk3(+/+) (0.4 +/- 0.1 micromol/24 h g body wt, 0.04 +/- 0.01%) mice. The sgk1(-/-)/sgk3(-/-) mice share the delayed hair growth with sgk3(-/-) mice and the modestly impaired renal salt retention with sgk1(-/-) mice. Additional lack of the isoform kinase does not substantially compound the phenotype for either property.