p53 Accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101−/− embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101−/− embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21WAF-1/CIP-1, was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101−/− embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.

Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

p63 identifies keratinocyte stem cells

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3σ has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

Indirect recognition of allopeptides promotes the development of cardiac allograft vasculopathy

Graft loss from chronic rejection has become the major obstacle to the long-term success of whole organ transplantation. In cardiac allografts, chronic rejection is manifested as a diffuse and accelerated form of arteriosclerosis, termed cardiac allograft vasculopathy. It has been suggested that T-cell recognition of processed alloantigens (allopeptides) presented by recipient antigen-presenting cells through the indirect pathway of allorecognition plays a critical role in the development and progression of chronic rejection. However, definitive preclinical evidence to support this hypothesis is lacking. To examine the role of indirect allorecognition in a clinically relevant large animal model of cardiac allograft vasculopathy, we immunized MHC inbred miniature swine with synthetic polymorphic peptides spanning the α1 domain of an allogeneic donor-derived swine leukocyte antigen class I gene. Pigs immunized with swine leukocyte antigen class I allopeptides showed in vitro proliferative responses and in vivo delayed-type hypersensitivity responses to the allogeneic peptides. Donor MHC class I disparate hearts transplanted into peptide-immunized cyclosporine-treated pigs not only rejected faster than unimmunized cyclosporine-treated controls (mean survival time = 5.5 +/−1.7 vs. 54.7 +/−3.8 days, P < 0.001), but they also developed obstructive fibroproliferative coronary artery lesions much earlier than unimmunized controls (<9 vs. >30 days). These results definitively link indirect allorecognition and cardiac allograft vasculopathy.

Peyer's patches are required for oral tolerance to proteins

To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treated in utero with lymphotoxin β-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Peyer's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4+ T-cell-derived IFN-γ, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.

Akt phosphorylates and regulates the orphan nuclear receptor Nur77

The immediate early gene NUR77 (also called NGFI-B) is required for T cell antigen receptor-mediated cell death and is induced to very high levels in immature thymocytes and T cell hybridomas undergoing apoptosis. The Akt (PKB) kinase is a key player in transduction of anti-apoptotic and proliferative signals in T cells. Because Nur77 has a putative Akt phosphorylation site at Ser-350, and phosphorylation of this residue is critical for the transactivation activity of Nur77, we investigated whether Akt regulates Nur77. Coimmunoprecipitation experiments showed the detection of Nur77 in Akt immune complexes, suggesting that Nur77 and Akt physically interact. We further show that Akt specifically phosphorylates Ser-350 of the Nur77 protein within its DNA-binding domain in vitro and in vivo in 293 and NIH 3T3 cells. Because phosphorylation of Ser-350 of Nur77 is critical for its function as a transcription factor, we examined the effect of Akt on this function. By using luciferase assay experiments, we showed that phosphorylation of Nur77 by Akt decreased the transcriptional activity of Nur77 by 50–85%. Thus, we show that Akt interacts with Nur77 and inactivates Nur77 by phosphorylation at Ser-350 in a phosphatidylinositol 3-kinase-dependent manner, connecting the phosphatidylinositol 3-kinase-dependent Akt pathway and a nuclear receptor pathway.

Selective ablation of retinoid X receptor α in hepatocytes impairs their lifespan and regenerative capacity

Retinoid X receptors (RXRs) are involved in a number of signaling pathways as heterodimeric partners of numerous nuclear receptors. Hepatocytes express high levels of the RXRα isotype, as well as several of its putative heterodimeric partners. Germ-line disruption (knockout) of RXRα has been shown to be lethal in utero, thus precluding analysis of its function at later life stages. Hepatocyte-specific disruption of RXRα during liver organogenesis has recently revealed that the presence of hepatocytes is not mandatory for the mouse, at least under normal mouse facility conditions, even though a number of metabolic events are impaired [Wan, Y.-J., et al. (2000) Mol. Cell. Biol. 20, 4436–4444]. However, it is unknown whether RXRα plays a role in the control of hepatocyte proliferation and lifespan. Here, we report a detailed analysis of the liver of mice in which RXRα was selectively ablated in adult hepatocytes by using the tamoxifen-inducible chimeric Cre recombinase system. Our results show that the lifespan of adult hepatocytes lacking RXRα is shorter than that of their wild-type counterparts, whereas proliferative hepatocytes of regenerating liver exhibit an even shorter lifespan. These lifespan shortenings are accompanied by increased polyploidy and multinuclearity. We conclude that RXRα plays important cell-autonomous function(s) in the mechanism(s) involved in the lifespan of hepatocytes and liver regeneration.

Distinct Cyclin D Genes Show Mitotic Accumulation or Constant Levels of Transcripts in Tobacco Bright Yellow-2 Cells1

The commitment of eukaryotic cells to division normally occurs during the G1 phase of the cell cycle. In mammals D-type cyclins regulate the progression of cells through G1 and therefore are important for both proliferative and developmental controls. Plant CycDs (D-type cyclin homologs) have been identified, but their precise function during the plant cell cycle is unknown. We have isolated three tobacco (Nicotiana tabacum) CycD cyclin cDNAs: two belong to the CycD3 class (Nicta;CycD3;1 and Nicta;CycD3;2) and the third to the CycD2 class (Nicta;CycD2;1). To uncouple their cell-cycle regulation from developmental control, we have used the highly synchronizable tobacco cultivar Bright Yellow-2 in a cell-suspension culture to characterize changes in CycD transcript levels during the cell cycle. In cells re-entering the cell cycle from stationary phase, CycD3;2 was induced in G1 but subsequently remained at a constant level in synchronous cells. This expression pattern is consistent with a role for CycD3;2, similar to mammalian D-type cyclins. In contrast, CycD2;1 and CycD3;1 transcripts accumulated during mitosis in synchronous cells, a pattern of expression not normally associated with D-type cyclins. This could suggest a novel role for plant D-type cyclins during mitosis.

T cell-specific FADD-deficient mice: FADD is required for early T cell development

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis of FADD−/− T cells from RAG-1−/− reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8− stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.

Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells

We describe a mouse model in which p27Kip1 transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18–26 h of transgene expression, the G1 phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G1 phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27Kip1 and control of G1 phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G1 phase length to a putative physiological maximum despite ongoing p27Kip1 transgene expression.

Biologic consequences of Stat1-independent IFN signaling

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.

Effects of estrogen on growth plate senescence and epiphyseal fusion

Estrogen is critical for epiphyseal fusion in both young men and women. In this study, we explored the cellular mechanisms by which estrogen causes this phenomenon. Juvenile ovariectomized female rabbits received either 70 μg/kg estradiol cypionate or vehicle i.m. once a week. Growth plates from the proximal tibia, distal tibia, and distal femur were analyzed after 2, 4, 6, or 8 weeks of treatment. In vehicle-treated animals, there was a gradual senescent decline in tibial growth rate, rate of chondrocyte proliferation, growth plate height, number of proliferative chondrocytes, number of hypertrophic chondrocytes, size of terminal hypertrophic chondrocytes, and column density. Estrogen treatment accelerated the senescent decline in all of these parameters. In senescent growth plates, epiphyseal fusion was observed to be an abrupt event in which all remaining chondrocytes were rapidly replaced by bone elements. Fusion occurred when the rate of chondrocyte proliferation approached zero. Estrogen caused this proliferative exhaustion and fusion to occur earlier. Our data suggest that (i) epiphyseal fusion is triggered when the proliferative potential of growth plate chondrocytes is exhausted; and (ii) estrogen does not induce growth plate ossification directly; instead, estrogen accelerates the programmed senescence of the growth plate, thus causing earlier proliferative exhaustion and consequently earlier fusion.

Clues to epidermal cancer proneness revealed by reconstruction of DNA repair-deficient xeroderma pigmentosum skin in vitro

Sun exposure has been clearly implicated in premature skin aging and neoplastic development. These features are exacerbated in patients with xeroderma pigmentosum (XP), a hereditary disease, the biochemical hallmark of which is a severe deficiency in the nucleotide excision repair of UV-induced DNA lesions. To develop an organotypic model of DNA repair deficiency, we have cultured several strains of primary XP keratinocytes and XP fibroblasts from skin biopsies of XP patients. XP skin comprising both a full-thickness epidermis and a dermal equivalent was succesfully reconstructed in vitro. Satisfactory features of stratification were obtained, but the expression of epidermal differentiation products, such as keratin K10 and loricrin, was delayed and reduced. In addition, the proliferation of XP keratinocytes was more rapid than that of normal keratinocytes. Moreover, increased deposition of cell attachment proteins, α-6 and β-1 integrins, was observed in the basement membrane zone, and β-1 integrin subunit, the expression of which is normally confined to basal keratinocytes, extended into several suprabasal cell layers. Most strikingly, the in vitro reconstructed XP skin displayed numerous proliferative epidermal invasions within dermal equivalents. Epidermal invasion and higher proliferation rate are reminiscent of early steps of neoplasia. Compared with normal skin, the DNA repair deficiency of in vitro reconstructed XP skin was documented by long-lasting persistence of UVB-induced DNA damage in all epidermal layers, including the basal layer from which carcinoma develops. The availability of in vitro reconstructed XP skin provides opportunities for research in the fields of photoaging, photocarcinogenesis, and tissue therapy.

Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotypic switch.

Leukemia inhibitory factor (LIF) promotes differentiated cell function in several systems. We recently reported LIF and LIF receptor expression in human fetal pituitary corticotrophs in vivo and demonstrated LIF stimulation of adrenocorticotrophin (ACTH) transcription in vitro, suggesting a role for LIF in corticotroph development. We therefore assessed the action of LIF on proliferating murine corticotroph cells (AtT20). LIF impairs proliferation of AtT20 cells (25% reduction versus control, P < 0.03), while simultaneously enhancing ACTH secretion (2-fold, P < 0.001) and augmenting ACTH responsiveness to corticotrophin-releasing hormone (CRH) action (4-fold, P < 0.001). This attenuation of cell growth is due to a block of cell cycle progression from G1 into S phase, as measured by flow cytometric analysis (24 +/- 0.8 versus 11.57 +/- 1.5, P < 0.001). Using bromodeoxyuridine incorporation assays, loss of cells in S phase was confirmed (25 +/- 0.08 to 9.4 +/- 1.4, P < 0.008). In contrast, CRH induced the G2/M phase (3.6 +/- 0.2 to 15.4 +/- 3, P < 0.001). This effect was blunted by LIF (P < 0.001 versus CRH alone). Cyclin A mRNA levels, which decline in S phase, were stimulated 3.5-fold by LIF and markedly suppressed by CRH. These results indicate a LIF-induced cell cycle block occurring at G1/S in corticotroph cells. Thus, LIF reduces proliferation, enhances ACTH secretion, and potentiates effects of CRH on ACTH secretion while blocking effects of CRH on the cell cycle. Responses of these three markers of differentiated corticotroph function indicate LIF to be a differentiation factor for pituitary corticotroph cells by preferential phenotypic switching from proliferative to synthetic.

Fusigenic viral liposome for gene therapy in cardiovascular diseases.

To improve the efficiency of liposome-mediated DNA transfer as a tool for gene therapy, we have developed a fusigenic liposome vector based on principles of viral cell fusion. The fusion proteins of hemagglutinating virus of Japan (HVJ; also Sendai virus) are complexed with liposomes that encapsulate oligodeoxynucleotide or plasmid DNA. Subsequent fusion of HVJ-liposomes with plasma membranes introduces the DNA directly into the cytoplasm. In addition, a DNA-binding nuclear protein is incorporated into the HVJ-liposome particle to enhance plasmid transgene expression. The fusigenic viral liposome vector has proven to be efficient for the intracellular introduction of oligodeoxynucleotide, as well as intact genes up to 100 kbp, both in vitro and in vivo. Many animal tissues have been found to be suitable targets for fusigenic viral liposome DNA transfer. In the cardiovascular system, we have documented successful cytostatic gene therapy in models of vascular proliferative disease using antisense oligodeoxynucleotides against cell cycle genes, double-stranded oligodeoxynucleotides as "decoys" to trap the transcription factor E2F, and expression of a transgene encoding the constitutive endothelial cell form of nitric oxide synthase. Similar strategies are also effective for the genetic engineering of vein grafts and for the treatment of a mouse model of immune-mediated glomerular disease.