CHOP is induced by fibrotic insult and promotes fibrosis.

<p>(A) CCl₄ injection promotes collagen deposition. Masson's trichrome staining was used to assess collagen deposition (blue) in formalin-fixed paraffin-embedded liver sections of mice injected i.p. with 10% CCl₄ twice a week for 12 weeks, or with mineral oil as a control. (B–C) CHOP expression is associated with CCl₄-challenged livers. (B) CHOP was detected by IHC in mouse livers from (A) and samples were counterstained with hematoxylin as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#pgen-1003937-g001" target="_blank">Figure 1C</a>. Representative data are shown. (C) The number of CHOP-positive nuclei per microscopic field from (B) was quantitated in multiple fields from 3 mice per group, and is expressed here as mean +/− S.E.M. per field. (D–E) <i>Chop</i> deletion attenuates DEN-induced fibrosis. Mild-to-moderate fibrosis is seen in wild-type but not <i>Chop</i>−/− livers by trichrome staining after DEN treatment as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#pgen-1003937-g002" target="_blank">Figure 2</a>. Sections from two separate DEN-treated animals of each genotype are shown. (E) A METAVIR score was blindly determined from trichrome stains as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#s4" target="_blank">Materials and Methods</a>, and the p-value was calculated by Asymptotic Wilcoxon Rank Sum Test. 0 = no fibrosis; 1 = portal fibrosis lacking septa; 2 = portal fibrosis with some septa; 3 = abundant septa; 4 = cirrhosis.</p

The Stress-Regulated Transcription Factor CHOP Promotes Hepatic Inflammatory Gene Expression, Fibrosis, and Oncogenesis

<div><p>Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. <i>Chop</i>-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. <i>Chop</i>-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.</p></div

CHOP is expressed in human HCC tumors.

<p>(A) Histology of tumor-proximal liver (uninvolved HCC) and tumor (two regions) tissue from the same patient. H&E images are representative of 28 tumor samples and 5 uninvolved matched controls. For (A) only, scale bar = 100 µm (B–C) CHOP immunostaining is associated with human HCC but not uninvolved regions. (B) Samples from uninvolved tumor-proximal tissue and HCC tumors were probed for CHOP expression by immunohistochemistry. CHOP-positive nuclei are indicated by arrowheads. Cellular and nuclear architecture in some HCC samples was distorted as seen in the non-counterstained image, accounting for the large nuclei. Scale bar = 50 µm. (C) The average number of CHOP-positive nuclei per microscopic field per patient was quantitated blindly from 5 HCC tumors and their uninvolved matched controls, as well as from 4 non-HCC specimens, and these were aggregated by Boxplot. P-values were calculated by Asymptotic Wilcoxon Rank Sum Test. (D) HCC-associated CHOP is expressed in hepatocytes. Expression of CHOP (brown) and cell type-specific markers (blue) for hepatocytes (cytokeratin-18), macrophages (CD68), bile ducts (cytokeratin-7), or activated stellate cells (smooth muscle actin) is shown by immunohistochemistry, with a hematoxylin counterstain applied. Representative data are shown. Note that only hepatocytes show CHOP-positive nuclei; arrowheads are used to show CHOP-positive cells in other samples. Scale bar = 50 µm. Insets show 25 µm×25 µm regions with distinct CHOP-positive nuclei.</p

CHOP promotes hepatocellular apoptosis and proliferation.

<p>(A) <i>Chop</i>−/− mice show reduced cell death upon DEN challenge. Apoptosis in vehicle- or DEN-treated livers of wild-type or <i>Chop</i>−/− mice was detected by IHC staining for the cleaved form of Caspase-3. Liver nodule borders are indicated by a dashed outline. Hematoxylin counterstain is not shown because it interfered with Caspase-3 staining. In (A–C), representative images are shown. (B–D) Reduced proliferation in <i>Chop</i>−/− DEN-challenged animals. Hepatocellular proliferation was detected by IHC staining for PCNA (B) or Ki-67 (C). (D) The extent of PCNA and Ki-67 staining was quantitated blindly as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003937#s4" target="_blank">Materials and Methods</a>. P-values were determined by Asymptotic Wilcoxon Rank Sum Test.</p

Microarray profiling reveals CHOP-dependent differences in inflammation and ribosomal biogenesis genes.

<p>(A) Basal expression differences in <i>Chop</i>−/− mice. 18,138 significantly expressed genes were analyzed using Illumina MouseRef-8 v2.0 BeadChips, with total RNA from 9 month old mouse liver homogenate as input. The number and percentage of genes upregulated (upper left) and downregulated (lower right) in wild-type versus <i>Chop</i>−/− tissue is shown, showing only genes differing by 1.5-fold or more, p<0.05. Expression is given on a log₂ scale. In (A), (D), and (E), the position of <i>Chop</i> is shown as a yellow-filled circle. (B–C) Suppressed basal expression of genes involved in immune and inflammatory pathways in <i>Chop</i>−/− mice. (B, top) GO pathways enriched among all regulated genes in PBS-treated wild-type versus <i>Chop</i>−/− animals were determined using DAVID, with all pathways that were significantly enriched after Bonferroni correction shown. (B, bottom) Same as (top), but considering only genes downregulated in <i>Chop</i>−/− animals. (C) Average array-determined expression of genotype-dependent immune- and inflammation-related genes from PBS-treated animals is shown on a log₂ scale +/− S.D.M. All genes shown are differentially expressed >1.5-fold (i.e., >0.585 on the y-axis), p<0.05. (D) DEN treatment alters gene expression in wild-type mice. Scatter plot of all genes differentially expressed (>1.5-fold; p<0.05) in tumors from 9 month-old DEN-treated wild-type animals versus normal liver tissue in age-matched PBS-treated wild-type animals. The positions of <i>Nfe2</i>, <i>Cyclin D1</i> (<i>Ccnd1</i>), and <i>β-catenin</i> are indicated by red diamonds. (E–F) Uniform upregulation of genes involved in ribosome biogenesis in tumors from <i>Chop</i>−/− mice. (E) Scatter plot of gene expression from tumor tissue of DEN-treated wild-type animals compared to <i>Chop</i>−/− animals is presented, showing all genes differing by >1.5-fold, p<0.05. (F) Average expression of all ribosomal subunit genes that were significantly different (p<0.05, fold-change >1.5) in <i>Chop</i>−/− versus wild-type large tumors is shown on a log₂ scale +/− S.D.M. as in (C).</p

CHOP is upregulated in a genetic mouse model of HCC.

<p>(A) eIF2α-dependent genes are upregulated in tumors induced by <i>SB</i> mutagenesis. Relative expression of the indicated UPR genes in tumors versus normal mouse liver tissue, as quantified by transcriptome sequencing. Sequencing was performed on total RNA from mouse liver tumors generated by T2/Onc3 transposition into the <i>Rtl1</i> locus (n = 8) and age-matched normal liver tissue (n = 7). The branch of UPR signaling to which each gene is most responsive is indicated. Here and in (B), error bars represent means +/− S.D.M. Here and elsewhere: *, p<0.05; **, p<0.01; ***, p<0.001. (B) qRT-PCR of <i>SB</i>-induced tumors confirms upregulation of <i>Chop</i>. Relative expression of the indicated genes quantitated by qRT-PCR from tumors arising from T2/Onc3 <i>Rtl1</i> locus integrants (n = 7), compared to expression in normal tissue (n = 5). Relative expression of total (tot) and spliced (spl) <i>Xbp1</i> mRNA is also shown. mRNA levels were normalized against average expression of 2 housekeeping genes (<i>Btf3</i> and <i>Ppia</i>). (C and D) CHOP and BiP expression, respectively, in tumors from <i>Rtl1</i> locus integrants and normal liver tissue from age-matched control animals using IHC. Also shown for CHOP staining are hematoxylin-counterstained samples, with exemplar CHOP-positive nuclei indicated by arrowheads. All scale bars throughout this work represent 50 µm unless indicated otherwise. The same anti-mouse immunoglobulin secondary antibody was used for both CHOP and BiP immunostains.</p

Model for the role of CHOP in HCC.

<p>The simplest interpretation of the data presented is a linear pathway whereby CHOP promotes apoptosis, which in turn activates inflammatory signaling, leading to fibrosis, compensatory proliferation, and ultimately tumorigenesis. Fibrosis and cellular transformation might in turn exacerbate cellular stress and CHOP expression, amplifying the tumor-promoting function of CHOP.</p

N-linked glycans are required for dSmo trafficking and activity.

<p><b>A.</b> dSmoNQ5 does not signal <i>in vitro</i>. Cl8 cells were transfected with control or <i>smo 5’UTR</i> dsRNA, the <i>ptcΔ136-luciferase</i> reporter, <i>pAc-renilla</i> control, <i>pAc-myc-smoWT</i> or <i>NQ5</i>, and <i>pAc-hh</i> or empty vector control. Hh-induced reporter activity (gray bars) was ablated by knockdown of endogenous <i>smo</i> and rescued by dSmo cDNA lacking UTR sequence for wild type, but not for dSmoNQ5. <b>B-B’</b>. dSmoNQ5 demonstrates altered sub-cellular localization. Cl8 cells expressing Calreticulin-EGFP-KDEL ER marker (GFP-ER, green) and Myc-SmoWT or NQ5 in the presence or absence of Hh were imaged by immunofluorescence microscopy. Wild type dSmo (anti-Myc, magenta) localized to puncta that did not overlap with the ER marker in the absence of Hh, and translocated to the plasma membrane in response to Hh. The NQ5 mutant overlapped with the ER marker under both conditions. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right box). <b>B’</b>. GFP-ER colocalizes with V5 tagged BiP, Calnexin (Cnx) and Calreticulin (Crc). DAPI marks the nucleus. <b>C-H.</b> dSmoNQ5 does not signal <i>in vivo</i>. Transgenes encoding wild type (G) or NQ5 (H) dSmo proteins were expressed in the <i>nubbin>dicer;smo</i>^{<i>3’UTR</i>} background (E). Whereas wild type Smo could rescue the loss of function phenotype induced by <i>smo</i>^{<i>3’UTR</i>}, dSmoNQ5 could not (G-H compared to F and C-D, control). <i>UAS-EGFP</i> was expressed in the <i>nubbin>dicer;smo</i>^{<i>3’UTR</i>} background and serves as a control for normalized transgene dosage (F). <b>I.</b> Wild type and NQ5 dSmo proteins are present at similar protein levels in wing imaginal disc tissue lysate. The dSmoN213Q,N336Q protein level is higher.</p

N213 glycosylation partially compensates for N336 N-glycan loss.

<p><b>A.</b> The indicated mutants were expressed in Cl8 cells. Each of the mutant proteins migrated more quickly in SDS-PAGE than wild type Smo, but not as quickly as SmoNQ4 or NQ5. <b>B.</b> Glycosylation at N213 partially compensates for N336 glycan loss. The rescue reporter assay was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2A</a>. Each of the indicated double mutants, with the exception of N213Q,N336Q, was able to rescue reporter gene expression in the <i>smo</i> knockdown background to a level similar to that of N336Q. dSmoN213Q,N336Q demonstrated a level of activity similar to dSmoNQ5. Significance was determined using Student’s t-test. <b>C.</b> N336Q-containing double mutants are retained in the ER. Cl8 cells expressing the indicated Smo proteins (anti-Myc, magenta), the Cal-EGFP-KDEL marker (green) and Hh were examined by immunofluorescence microscopy. Whereas wild type Smo reached the plasma membrane, the double mutants overlapped with the ER marker. ActinRed (red) marks F-actin. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). <b>D.</b> Treatment of lysates from WT or N213Q,N336Q expressing cells with deglycosylating enzymes reveals that the N213,336Q mutant is present in the EndoH sensitive ER fraction, arrowhead. <b>E-E’</b>. N336Q and N213Q,N336Q mutants have disulfide bond defects. Biotin-maleimide was used to tag free thiol groups in cellular lysates prepared from Cl8 cells expressing WT, N336Q, N213Q,N336Q and C320A dSmo proteins. WT dSmo is not well captured on NeutrAvadin beads (lane 2, bound). N-glycan mutants are captured similarly to the disulfide bond mutant C320A (lanes 3–5, bound), indicating that at least one disulfide bridge is disrupted by N-glycan loss. E’ shows the ratio of bound to unbound dSmo proteins normalized to kinesin. Relative binding was determined by densitometry analysis of two independent binding assays. C320A, which has an established disulfide bond defect served as positive control. It’s binding ratio was arbitrarily set to 1.0 and other values are shown relative to it. Error bars are provided to show the standard deviation between the two experiments. <b>F.</b> dSmoN213Q,N336Q fails to rescue <i>smo</i> knockdown <i>in vivo</i>. <i>UAS-dsmoN213Q</i>, <i>N336Q</i> was co-expressed with <i>UAS-dicer</i> and <i>UAS-smo</i>^{<i>3’UTR</i>} using the <i>nubbin-Gal4</i> driver. Its expression did not modify the <i>smo</i> knockdown phenotype (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2F</a>). Multiple progeny were analyzed over two crosses and a representative wing is shown.</p

Identification of Smo N-linked glycosylation sites.

<p><b>A</b>. A multiple sequence alignment of Smo proteins from different phyla are shown. Consensus sequences for N-linked glycosylation are highlighted in gray and the Asn acceptor residues are bold. Sites conserved across vertebrate proteins are indicated as N1-N4. The predicted <i>D</i>. <i>melanogaster</i> sites are not tightly conserved with vertebrates. <b>B</b>. Drosophila Smo is N-glycosylated. Cell lysates prepared from Cl8 cells expressing Hh with wild type or NQ5 dSmo proteins were treated with the indicated deglycosylating enzymes. Wild type Smo demonstrated ER (arrow) and post-ER (arrowhead) glycosylation species. NQ5 migrated similarly to the fully deglycosylated species under all conditions (arrow). <b>C</b>. Mouse Smo is N-glycosylated. Cellular lysates from <i>Smo-/-</i> cells stably expressing mSmoWT or mSmoNQ4 were treated with deglycosylating enzymes and subjected to SDS-PAGE and western blot. mSmoWT resolves as two distinct forms (lane 2). The arrow marks the ER form and the arrowhead indicates the post-ER form. mSmoNQ4 migrates in SDS-PAGE similarly to the PNGase-treated wild type protein (lanes 4–5). <b>C’</b>. mSmoNQ4 is O-glycosylated. Lysates were prepared from NIH3T3 cells expressing mSmoNQ4 and subjected to lambda phosphatase, PNGase and O-glycosidase/neuraminidase treatments. The upper band collapsed upon O-glycosidase/neuraminidase treatment. <b>D</b>. Expression of individual N to Q dSmo mutants. The indicated N to Q dSmo mutants were expressed in Cl8 cells and cell lysates were analyzed by SDS-PAGE and western blot against the Myc tag. Kin serves as loading control. <b>E</b>. Extracellular mSmo consensus sites are N-glycosylated. Mutation of individual extracellular mSmo glycosylation sites induced faster mobility on SDS-PAGE. mSmoN450Q migrated similarly to SmoWT. For western blots, mSmo was detected using anti-Smo and dSmo with anti-Myc. Kinesin (Kin) and Tubulin (Tub) were blotted for loading controls.</p