LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus

The LIM domain protein LPP is a coactivator for the ETS domain transcription factor PEA3

PEA3 is a member of a subfamily of ETS domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression

Zfp148 deficiency causes lung maturation defects and lethality in newborn mice that are rescued by deletion of p53 or antioxidant treatment.

The transcription factor Zfp148 (Zbp-89, BFCOL, BERF1, htβ) interacts physically with the tumor suppressor p53 and is implicated in cell cycle control, but the physiological role of Zfp148 remains unknown. Here we show that Zfp148 deficiency leads to respiratory distress and lethality in newborn mice. Zfp148 deficiency prevented structural maturation of the prenatal lung without affecting type II cell differentiation or surfactant production. BrdU analyses revealed that Zfp148 deficiency caused proliferation arrest of pulmonary cells at E18.5-19.5. Similarly, Zfp148-deficient fibroblasts exhibited proliferative arrest that was dependent on p53, raising the possibility that cell stress is part of the underlying mechanism. Indeed, Zfp148 deficiency lowered the threshold for activation of p53 under oxidative conditions. Moreover, both in vivo and cellular phenotypes were rescued on Trp53(+/-) or Trp53(-/-) backgrounds and by antioxidant treatment. Thus, Zfp148 prevents respiratory distress and lethality in newborn mice by attenuating oxidative stress-dependent p53-activity during the saccular stage of lung development. Our results establish Zfp148 as a novel player in mammalian lung maturation and demonstrate that Zfp148 is critical for cell cycle progression in vivo

Deletion of one or two copies of <i>Trp53</i> rescued <i>Zfp148^gt/gt</i> mice from proliferation arrest, respiratory distress and neonatal lethality.

<p>(A) Photomicrographs showing BrdU labeling of E19.5 lung from wt, <i>Zfp148^gt/gt</i>, <i>Zfp148^gt/gtTrp53^+/−</i> and <i>Trp53^+/−</i> mice. Graphs show quantification of BrdU positive cells per lung area (<i>n</i> = 6 wt, 7 <i>Zfp148^+/gt</i>, 7 <i>Zfp148^gt/gt</i>, 5 <i>Zfp148^gt/gtTrp53^+/−</i>, 4 <i>Trp53^+/−</i>). (B) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from <i>Zfp148^+/+Trp53^+/+</i> (<i>n</i> = 10), <i>Zfp148^gt/gtTrp53^+/+</i> (<i>n</i> = 9), <i>Zfp148^gt/gtTrp53^+/−</i> (<i>n</i> = 9), <i>Zfp148^gt/gtTrp53^−/−</i> (<i>n</i> = 5), and <i>Zfp148^+/+Trp53^−/−</i> (<i>n</i> = 10) mice, respectively. Graphs show quantification of mean tissue area per total lung area, PAS-positive area with bronchioles excluded, and CC10-positive area with bronchioles excluded. (C) Distribution of <i>Zfp148</i> genotypes of P1 pups of intercrosses between <i>Zfp148^+/gtTrp53^+/+</i> and <i>Zfp148^+/gtTrp53^+/−</i> mice. Scale bars, 100 µm. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Antioxidant rescue of defect lung maturation and neonatal lethality in <i>Zfp148^gt/gt</i> mice.

<p>(<b>A</b>) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from <i>Zfp148^+/+</i>, <i>Zfp148^gt/gt</i>, and NAC treated <i>Zfp148^gt/gt</i> mice, respectively (<i>n</i> = 6). Graphs show quantification of mean tissue area per total lung area, PAS-positive area with bronchioles excluded, and CC10-positive area with bronchioles excluded. (<b>B</b>) Distribution of <i>Zfp148</i> genotypes of P1 pups of intercrosses between <i>Zfp148^+/gt</i> mice with and without NAC treatment. Scale bars, 100 µm. **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>Zfp148</i> deficiency prevented structural maturation of prenatal lungs without effecting epithelial cell differentiation or surfactant production.

<p>(<b>A</b>) Photomicrographs showing lung morphology (hematoxylin and eosin; HE) and glycogen content (periodic acid-Shiff; PAS) and CC10 immunofluorescence in P1 lungs from <i>Zfp148^gt/gt</i> and wt mice, respectively. (<b>A, B</b>) Graphs show quantification (<i>n</i> = 6) of mean tissue area per total lung area, PAS-positive area with bronchioles excluded, and CC10-positive area with bronchioles excluded in (A) P1 and (B) E19.5 lungs from wt and <i>Zfp148^gt/gt</i> mice. a.u., arbitrary units. (<b>C</b>) Real-time RT-PCR showing relative expression levels of markers for type I (T1alpha, Aqp5), type II (Sftpa1, Sftpb, Sftpc, Sftpd), clara (CC-10, Pon1), endothelial (Pecam1, Tie2, Nos3) and smooth muscle (Acta2) cells in <i>Zfp148^gt/gt</i> lungs compared to wt at P1 (<i>n</i> = 6). Wt means are represented by the horizontal straight line at 1. (<b>D</b>) Transmission electron microscope (TEM) image of <i>Zfp148^gt/gt</i> lung at E18.5–19.5 showing lamellar bodies secreted into the lumen of a terminal sac. (<b>E–G</b>) TEM images showing differentiated cells in <i>Zfp148^gt/gt</i> lungs at E18.5–19.5. (E) Apical part of an alveolar type II cell containing typical lamellar bodies (arrowheads), one of which is in the process of exocytosis (arrow), and accumulations of densely contrasted glycogen particles (asterisk). (F) Two ciliated cells (arrowheads) surrounding two Clara cells (arrows) with bulging appearance and mitochondria accumulated in the apical cytoplasm. (G) High power view of the blood-alveolar barrier showing an erythrocyte (asterisk) in close contact with a highly attenuated part of an endothelial cell that shares a basal lamina with the alveolar type I cell. Scale bars, 2 µm. *<i>P</i><0.05, ***<i>P</i><0.001.</p

<i>Zfp148</i> deficiency causes lethality in newborn mice and growth retardation and reduced life span in adult mice.

<p>(<b>A</b>) <i>Zfp148</i>-genotype distribution of offspring from heterozygous intercrosses. (<b>B</b>) Photograph of cyanotic <i>Zfp148^gt/gt</i> mouse and wt littermate at P1. (<b>C</b>) Body weight of wt, <i>Zfp148^+/gt</i> and <i>Zfp148^gt/gt</i> mice of mixed genders at E18.5–19.5 (<i>n</i> = 12 wt, 15 <i>Zfp148^+/gt</i>, 8 <i>Zfp148^gt/gt</i>), P1 (<i>n</i> = 9 wt, 13 <i>Zfp148^+/gt</i>, 7 <i>Zfp148^gt/gt</i>), P9 (<i>n</i> = 28 wt, 44 <i>Zfp148^+/gt</i>, 10 <i>Zfp148^gt/gt</i>) and P19–22 (<i>n</i> = 10 wt, 18 <i>Zfp148^+/gt</i>, 9 <i>Zfp148^gt/gt</i>). (<b>D, E</b>) Body weight curves for adult wt and <i>Zfp148^gt/gt</i> male (D) and female (E) mice, respectively (<i>n</i> = 10). (<b>F</b>) Kaplan-Meier plots showing survival of <i>Zfp148^gt/gt</i> and wt mice (<i>n</i> = 20). ***<i>P</i><0.001.</p

Activation of p53 and <i>Trp53</i>-dependent proliferation arrest in <i>Zfp148^gt/gt</i> MEFs.

<p>(<b>A</b>) Western blots of MEF lysates showing expression of phospho-p53^Ser18 in wt and <i>Zfp148^gt/gt</i> MEFs cultured at 21 or 3% oxygen, respectively. <i>Trp53^−/−</i> cells were used as a negative control and actin was used as a loading control. Asterisk indicates unspecific band. (<b>B</b>) Real-time RT-PCR of <i>p21</i> in wt and <i>Zfp148^gt/gt</i> MEFs cultured at 21 or 3% oxygen, respectively, and on <i>Trp53^+/+</i>, <i>Trp53^+/−</i> and <i>Trp53^−/−</i> genetic backgrounds (<i>n</i> = 4). (<b>C, D</b>) CPD of <i>Zfp148^gt/gt</i> and wt MEFs on <i>Trp53^+/+</i> or <i>Trp53^−/−</i> (C) and <i>Trp53^+/+</i> or <i>Trp53^+/−</i> (D) genetic backgrounds (<i>n</i> = 3). (<b>E, F</b>) CPD of <i>Zfp148^gt/gt</i> and wt MEFs supplemented with <i>n</i>-acetyl-L-cysteine (NAC) (E) in the culture medium (<i>n</i> = 4), or cultured at atmospheric (21%) or low (3%) oxygen concentrations (F) (<i>n</i> = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p