<i>In Vivo</i> Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

<div><p>Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in <i>SBDS</i> are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15^Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.</p></div

Key haplotypes in <i>MSRA</i> region showing association with MI in TSS cohort.

<p><b>^a</b>Numbers in parentheses indicate physical position on chr8 in base pairs (NCBI Build 36).</p><p><b>^b</b>All observed haplotypes with frequency >1% are shown; bold indicates haplotypes noted in text.</p><p>*Protective haplotypes that reached statistical significance after Bonferroni correction for all 2,890 haplotypes tested (<i>P</i><1.73×10⁻⁵).</p>‡<p>Near significant risk haplotype containing rs614197 (italicized), the SNP that was most highly associated with MI in the initial analysis.</p

Kaplan-Meier survival curves in CF mice according to <i>Msra</i> genotype.

<p>A. CF mice homozygous for a null <i>Cftr</i> allele (<i>Cftr</i>^−/−) and wild-type for <i>Msra</i> show high mortality due to intestinal obstruction around the time of weaning (ca. 21 days; n = 30). In contrast, survival is markedly improved in <i>Cftr</i>^−/− mice lacking one (n = 33) or two <i>Msra</i> alleles (n = 46) compared to wild-type (<i>P</i> = 0.022 and <i>P</i> = 0.0001, respectively; log-rank test). B. CF mice homozygous for a missense <i>Cftr</i> allele (<i>Cftr</i>^R117H/R117H) and wild-type for <i>Msra</i> display a low rate of mortality due to intestinal obstruction around the time of weaning (n = 14). Survival is not affected in <i>Cftr</i>^R117H/R117H mice lacking one (n = 51) or two (n = 51) <i>Msra</i> alleles compared to wild-type.</p

Regional association of SNPs within a region of linkage to MI on chromosome 8p23.1.

<p>The inset plot shows the locus linked to MI on chromosome 8 identified by Blackman, et al <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002580#pgen.1002580-Blackman1" target="_blank">[12]</a>. The green shaded region under the peak extending from 17.9 cM (8.2 Mb) to 29.0 cM (17.2 Mb), where LOD score >1, indicates the region analyzed in the main plot. The map position of <i>MSRA</i> is denoted by an arrow at ∼20.2 cM. In the main plot, <i>P</i> values are plotted in log scale versus physical location in Mb. The SNP showing the strongest association with MI, rs614197, is represented by a diamond (<i>P</i> = 8.35×10⁻⁶). SNPs surrounding rs614197 are color coded to reflect their LD with this SNP (pair-wise r² using 1000 Genomes CEU, August 2009). The dashed line indicates the threshold for region-wide significance after Bonferroni correction for 2,896 SNPs (P<1.73×10⁻⁵). Genes, exon positions, and direction of transcription are denoted below plot (human genome build 18). Nine genes outside this interval were omitted for display purposes: <i>C8orf12</i>, <i>FAM167A</i>, <i>DEFB136</i>, <i>DEFB135</i>, <i>DEFB134</i>, <i>FAM66D</i>, <i>LOC392196</i>, <i>USP17L2</i>, and <i>FAM86B1</i>. The blue shaded region represents the 2 Mb encompassing rs614197 in which haplotype association was tested (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002580#pgen.1002580.s001" target="_blank">Figure S1</a>).</p

Patient characteristics.

<p><b>^a</b>Primary analysis included subjects from 133 “MI families” in which at least one sibling had MI.</p><p><b>^b</b><i>CFTR</i> mutation-specific analysis (i.e. p.Gly551Asp vs. p.Phe508del) utilized the entire TSS sample.</p

Haplotype association in <i>MSRA</i> region.

<p>In this schematic, haplotypes comprised of three consecutive SNPs are represented as dots connected by a line. Two overlapping haplotypes localized within intron 3 of <i>MSRA</i> had a statistically significant protective effect on MI (<i>P</i><1.73×10⁻⁵ after Bonferroni correction for 2,890 haplotypes tested in a 2 Mb region): rs10903323 T – rs4840475 G – rs17151637 A and rs4840475 G – rs17151637 A – rs6601427 C. A “risk” haplotype upstream of <i>MSRA</i>, rs586123 G – rs614197 G – rs2055729 C, was just below the threshold for significant association. Linkage disequilibrium patterns (pairwise r²) are displayed below the rs numbers corresponding to each SNP (physical location provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002580#pgen-1002580-t002" target="_blank">Table 2</a>).</p

Tgfβ and p53 response in the SDS pancreas.

<p><b>A,</b> Steady state protein levels paralleled observed transcript changes with decreased Myc and increased p21^Cip and Tgfβ expression, along with changes in Smad2 and Smad3 phosphorylation status in mutants. Representative immunoblots of lysates from four littermate pairs at 3 weeks of age are shown. Associated densitometry is shown in right graphs, with Myc, Tgfβ, and p21 relative to Gapdh expression, and phosphorylated-Smad2 and Smad3 relative to total Smad2 and Smad3, respectively. Horizontal lines in scatter plots indicate mean values. <b>B,</b> Representative immunoblot indicates stabilization of p53 protein in the SDS pancreas at 3 weeks of age with associated densitometry (expression relative to calnexin) below. Horizontal lines indicate mean values. <b>C,</b> Immunohistochemistry indicated p53 stabilization as early as 15 days of age (sections shown are of littermates). Yellow arrows highlight examples of positive nuclei. p53 staining was observed specifically in nuclei of acinar cells of the SDS pancreas model (islets denoted with pale yellow dashed outlines). Scale bars represent 50 μm.</p

SDS brain is apoptotic.

<p>Histochemistry of transverse rhombencephalon (E11.5; <b>A</b>) and telencephalon (E14.5; <b>B</b>) brain sections indicate hypocellularity and neuronal cell death (green in TUNEL panels) in post-mitotic regions of <i>Sbds</i>^{<i>R126T/R126T</i>} mice with earlier onset in the <i>Sbds</i>^{<i>R126T/–</i>}mice (compare TUNEL panels in <b>A</b> and <b>B</b>). Bromodeoxyuridine labeling (brown in BrdU panels) highlighted reduced proliferation of neural progenitors. V, lateral ventricle; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars represent 25 μm.</p

Senescence-associated markers in the SDS pancreas.

<p><b>A,</b> Senescence-associated β-galactosidase activity (SA-bgal, bright blue) was detected in acini of the SDS pancreas at 30 days of age (N = 5). <i>Sbds</i>^{<i>P–/+</i>} and <i>Sbds</i>^{<i>P–/R126T</i>} are shorthand for <i>Sbds</i>^<i>CKO/+</i>; <i>Ptf1a</i>^<i>Cre/+</i> and <i>Sbds</i>^{<i>CKO/R126T</i>}; <i>Ptf1a</i>^<i>Cre/+</i>, respectively [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005288#pgen.1005288.ref051" target="_blank">51</a>]. Scale bars represent 100 μm. <b>B,</b> 84 cellular-senescence associated genes were assayed using the SABiosciences Cellular Senescence RT² Profiler PCR Array (QIAGEN) with total RNA isolated from pancreata of mice at 15 and 25 days of age. Table lists transcripts that showed statistically significant changes relative to control genes at at least one of the two assayed time points (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005288#pgen.1005288.s010" target="_blank">S2 Table</a>). Fold change: <i>Sbds</i>^{<i>P-/R126T</i>}/<i>Sbds</i>^<i>P-/+</i>. Criteria for significance (as per supplier’s instructions): ≥3 fold difference with a <i>P</i>-value of <0.05, N = 3 at each time point. <b>C</b> and <b>D,</b> Quantitative transcript analysis. In <b>C</b>, fold change: <i>Sbds</i>^{<i>P-/R126T</i>}/<i>Sbds</i>^<i>P-/+</i>; N = 4 at each time point, except at E18.5 where N = 3. Criteria for significance: ≥2 fold change, <i>P</i><0.05. E18.5 pancreas expression is relative to Tbp; P8-P25 expression is relative to Gapdh. In <b>D</b>, fold change: <i>Sbds</i>^{<i>R126T/R126T</i>}/<i>Sbds</i>^{<i>R126T/+</i>}; N = 4. Criteria for significance: ≥2 fold change, <i>P</i><0.05. Brain and liver expression is relative to Actb; bone expression is relative to Tbp. All <i>P</i>-values calculated using unpaired, two-tailed T-tests. Red indicates down-regulation, blue indicates up-regulation. Abbreviations in <b>B</b>: <i>Akt1</i>: <i>Thymoma viral proto-oncogene 1; Cd44</i>: <i>CD44 antigen; Cdkn1a</i>: <i>Cyclin-dependent kinase inhibitor 1A; Ckdn2b</i>: <i>Cyclin-dependent kinase inhibitor 2B; Col1a1</i>: <i>Collagen</i>, <i>type I</i>, <i>alpha 1; Creg1</i>: <i>Cellular repressor of E1A-stimulated genes 1; Egr1</i>: <i>Early growth response 1; Ets1</i>: <i>E26 avian leukemia oncogene 1</i>, <i>5’ domain; Fn1</i>: <i>Fibronectin 1; Hras1</i>: <i>Harvey rat sarcoma virus oncogene 1; Ifng</i>: <i>Interferon gamma; Igfbp5</i>: <i>Insulin-like growth factor binding protein 5; Igfbp7</i>: <i>Insulin-like growth factor binding protein 7; Interferon regulatory factor 3; Irf5</i>: <i>Interferon regulatory factor 5; Irf7</i>: <i>Interferon regulatory factor 7; Myc</i>: <i>Myelocytomatosis oncogene; Nfkb1</i>: <i>Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1</i>, <i>p105; Sparc</i>: <i>Secreted acidic cysteine rich glycoprotein (osteonectin; Tbx3</i>: <i>T-box-3; Tgfb1</i>: <i>Transforming growth factor</i>, <i>beta 1; Tgfb1i1</i>: <i>Transforming growth factor beta 1 induced transcript 1</i>.</p

Sbds mutants display ribosomopathy and SDS phenotypes.

<p><b>A,</b> Embryos with biallelic mutations in <i>Sbds</i> have decreased mass compared with littermate controls, **<i>P</i><4X10^-6. <i>Sbds</i>^{<i>R126T/–</i>}embryos are smaller than <i>Sbds</i>^{<i>R126T/R126T</i>} embryos, *<i>P</i> = 1.9X10^-4 (Wilcoxon Rank Sum Test; Kruskal-Wallis <i>P</i> = 3.0X10^-8). Error bars represent ±SEM. Scale bar represents 5 mm (upper panel). <b>B,</b> Decreased granulocytes (dark purple, H&E, E18.5) in liver (cell cluster examples are indicated with yellow arrowheads) and bone marrow (black arrowheads) with loss of <i>Sbds</i>; N = 3 (<i>Sbds</i>^{<i>R126T/R126T</i>}) and 4 (<i>Sbds</i>^{<i>R126T/–</i>}). Scale bars represent 100 μm. <b>C,</b> Decreased bone ossification was observed in transverse metacarpal sections of mutants (corresponding regions of littermate controls that maintain red Safranin O staining in mutants are highlighted with magenta arrowheads, E18.5). Scale bars represent 100 μm.</p