Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX
Address reprint requests to: Robert Y.L. Tsai, M.D., Ph.D., Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030. E-mail: email@example.com; fax: 713-677-7512.
Potential conflict of interest: Nothing to report.
This work was supported, in part, by a Texas A&M Cancer Research Council Incentive Award (to R.Y.L.T.), a National Institutes of Health (NIH) grant (P30 DK56338) that supports the Texas Medical Center Digestive Diseases Center (to R.Y.L.T. and M.J.F.), and an Egyptian Cultural and Educational Bureau postdoctoral fellowship (to W.I.). The authors thank Dr. Valentina Factor (National Cancer Institute, NIH, Bethesda, MD) for providing A6 antibody.
During liver development and regeneration, hepatocytes undergo rapid cell division and face an increased risk of DNA damage associated with active DNA replication. The mechanism that protects proliferating hepatocytes from replication-induced DNA damage remains unclear. Nucleostemin (NS) is known to be up-regulated during liver regeneration, and loss of NS is associated with increased DNA damage in cancer cells. To determine whether NS is involved in protecting the genome integrity of proliferating hepatocytes, we created an albumin promoter-driven NS conditional-null (albNScko) mouse model. Livers of albNScko mice begin to show loss of NS in developing hepatocytes from the first postnatal week and increased DNA damage and hepatocellular injury at 1-2 weeks of age. At 3-4 weeks, albNScko livers develop bile duct hyperplasia and show increased apoptotic cells, necrosis, regenerative nodules, and evidence suggestive of hepatic stem/progenitor cell activation. CCl4 treatment enhances degeneration and DNA damage in NS-deleted hepatocytes and increases biliary hyperplasia and A6+ cells in albNScko livers. After 70% partial hepatectomy, albNScko livers show increased DNA damage in parallel with a blunted and prolonged regenerative response. The DNA damage in NS-depleted hepatocytes is explained by the impaired recruitment of a core DNA repair enzyme, RAD51, to replication-induced DNA damage foci. Conclusion: This work reveals a novel genome-protective role of NS in developing and regenerating hepatocytes. (Hepatology 2013; 58:2176–2187)
Fetal hepatoblasts and hepatic progenitors undergo rapid proliferation during liver organogenesis. After maturation, hepatocytes become mitotically inactive, but retain the ability to reenter the cell cycle after acute liver injuries. Subsequent to massive damage, adult livers also recruit hepatic stem/progenitor cells (HSPCs) as a source for regeneration. During the process of DNA replication, spontaneous damage may occur as a result of stalled and collapsed replication forks.[1, 2] To date, it remains unclear how HSPCs and proliferative hepatocytes avoid the increased risk of replication-induced DNA damage and whether molecules beyond the core DNA damage-repair machinery help protect the integrity of their genome.[3, 4]
Nucleostemin (NS) was discovered first in neural stem cells and later in other types of stem/progenitor cells and cancers.[5, 6] The biological significance of NS is exemplified by the early embryonic lethal phenotype of germline NS-knockout (NSKO) mice. Its importance in stem cells is shown by NS-knockdown (NSKD)-induced self-renewal impairment and the ability of NS to promote pluripotency in conjunction with SRY (sex determining region Y)-box 2 (Sox2) and octamer-binding transcription factor 4.[8, 9] In adult animals, the expression of NS is low in most tissues, except for the testis, but is up-regulated during regeneration in several tissues.[10, 11] A potential role of NS in liver biology is indicated by a recent study showing an increased expression of NS in hepatic precursor cells and adult livers after partial hepatectomy.
Because loss of NS increases spontaneous DNA damage in cancer cells, we hypothesize that it may have a role in protecting the genome integrity of actively dividing HSPCs and hepatocytes. To test this idea, we created a hepatocyte-specific NS conditional-knockout (albNScko) mouse model by introducing an albumin promoter-driven Cre transgene (Alb-Cre) into a new NS-flox (NSflx) mouse model. Because the albumin promoter gets turned on gradually during gestation and postnatal development, we anticipate that the albNScko model will abolish the activity of NS in the Alb+ differentiating hepatocytic progenitors and regenerating hepatocytes and allow us to address its role in liver development and regeneration. Indeed, livers of albNScko mice show early-onset DNA damage at 2 weeks, followed by an increase of apoptotic cells, regenerative nodules, and biliary hyperplasia at 3-4 weeks. In response to CCl4-induced damage or 70% partial hepatectomy (PHx), albNScko livers show enhanced degeneration and DNA damage in NS-deleted hepatocytes. Mechanistically, loss of NS triggers replication-dependent DNA damage by reducing recruitment of RAD51 to hydroxyurea (HU)-induced damage foci. These data establish a novel role of NS in protecting the genomic stability of dividing hepatocytes.
Materials and Methods
Animals were handled in accord with the Guide for the Care and Use of Laboratory Animals, and procedures were approved by the institutional animal care and use committee. For acute CCl4 treatment, mice were injected intraperitoneally once with CCl4 (10 uL/g body weight). Oil- and CCl4-injected littermates were separately raised postinjection. To perform PHx, mice were anesthetized by inhalation of isoflurane. The left lateral and medium lobe of the liver were ligated and removed.
Creation of albNScko Mice
AlbNScko mice were created by crossing Alb-Cre transgenic mice with NSflx mice. NSflx mice were generated by the targeting strategy outlined in Supporting Fig. 1. Correctly targeted ES clones were identified by southern blottings. NSflxneo heterozygotes were mated with Rosa26flp mice to remove the pgk-neo cassette and generate NSflx heterozygotes.
Tissue Preparation, Immunohistochemistry, and TdT-Mediated dUTP Nick End Labeling Assay
Liver samples were fixed in HistoChoice (AMRESCO LLC, Solon, OH) and embedded in paraffin for hematoxylin and eosin (H&E), Sirius Red, anti-NS (Ab2438; 1-year-old livers), anti-cytokeratin 19 (CK19; TROMA-III; Developmental Studies Hybridoma Bank, Iowa City, IA), A6 (provided by Dr. Valentina Factor, National Cancer Institute, National Institutes of Health, Bethesda, MD), anti-γ-H2AX (JBW301; Upstate Biotechnology, Lake Placid, NY), anti-bromodeoxyuridine (BrdU; BU1/75; Accurate Chemical & Scientific Corporation, Westbury, NY), anti-alpha-fetoprotein (AFP; Biocare Medical, Concord, CA), anti-albumin (Novus Biologicals, LLC, Littleton, CO), anti-Sox9 (Millipore, Billerica, MA), anti-cytochrome P450 (CYP)2E1 (Millipore), and TdT-mediated dUTP nick end labeling (TUNEL) staining. For NS staining (Ab2438) in developing livers, fresh-frozen samples were collected and postfixed in 10% formalin. The specificity of Ab2438 was validated previously[7, 17] and in Supporting Fig. 2H. Apoptotic cells were labeled by the Deadend Fluorometric TUNEL system (Promega, Madison, WI). Nuclei were counterstained by TO-PRO®-3 (ToPro-3; Invitrogen, Carlsbad, CA).
Hepatocyte Culture, Transfection, Knockdown, and DNA Damage Analysis
For details on hepatocyte culture, transfection, knockdown, and DNA damage analysis, see the Supporting Data.
Data were presented as mean ± standard error of the mean (SEM). Differences between groups were analyzed by 2-tailed Student t test and considered as statistically significant when P values were less than 0.05.
Alb-Cre-Driven NS Deletion Causes Liver Damage Associated With Biliary Hyperplasia
To determine the role of NS in liver regeneration, we injected 8-week-old mice with CCl4. Northern blottings showed that the expression level of NS was relatively low in uninjured livers (Ctrl), but began to increase shortly after CCl4 injection (Fig. 1A, top). NS up-regulation peaks in 1 day and declines rapidly after 2 days, whereas the peak increase of BrdU-labeled cells occurs 2 days after injection (Fig. 1A, bottom). We created an NSflx model and showed that homozygous NSflx mice developed and grew normally, and homozygous deletion of the floxed sequence by a germline Cre transgene caused early embryonic lethality at E3.5 (n = 110; Fig. 1B and Supporting Fig. 1). To address the functional importance of NS in developing hepatocytes, we generated the albNScko mouse model by breeding the Alb-Cre transgene into NSflx/flx mice. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assays confirmed that NS expression was significantly reduced in albNScko livers from 1-4 weeks of age (Fig. 1C, top). Cre expression was found only in albNScko livers and showed a prominent peak at 2 weeks of age (Fig. 1C, bottom). Histologically, albNScko livers appeared no different from NSflx/flx livers up to 1 week of age, but began to show increased cellularity around bile ducts at 2 weeks of age (Supporting Fig. 2A-D). When albNScko mice reached 3-4 weeks of age, the liver surface displayed a nodular appearance (Fig. 1D) and showed areas of extensive bile duct hyperplasia (BDH; Fig. 1E1,E2 and Supporting Fig. 2E,F), portal and periportal fibrosis (Supporting Fig. 2G), and necrotic foci in parenchyma (Fig. 1E3). The liver/body weight ratio of albNScko mice began to exceed that of NSflx/flx mice at 3 weeks (Fig. 1F). These results demonstrate that NS deletion caused liver parenchymal damage and BDH.
DNA Damage Is an Early Event of albNScko, Followed by Apoptosis and Hepatic Regeneration
To establish onset of NS deletion and cell type(s) involved, we examined NS expression in albNScko livers from postnatal day 1 (P1D) to 3 weeks of age. Though a significant number of albNScko hepatocytes still retained their NS expression at P1D, almost all of them lost their NS expression at 1 and 2 weeks of age (Fig. 2A). These findings are consistent with a previous report that differentiated hepatocytes functionally expressing Alb-Cre are rare and distribute in a mosaic pattern in fetal livers. In 2- to 3-week-old albNScko livers, NS-positive cells were mostly confined to the hyperplastic ductular epithelium (Fig. 2B, left panels) and the regenerative hepatic nodules (Fig. 3D1). Contrarily, in the 3-week-old NSflx/flx liver, NS signals were found only in scattered hepatocytes, but not in bile duct epithelial cells (BECs; Fig. 2B, right panels). Although we cannot exclude the expression of Alb-Cre in subsets of BECs, these results indicate that expression of NS is depleted predominantly in the hepatocytic lineage by albNScko from 1 week of age, but is maintained in the hyperplastic BECs.
The time sequence of NSKO-induced events was determined by measuring onset of DNA damage, cell death, and hepatic regeneration in albNScko livers. DNA damage in vivo was detected by the foci formation of phosphorylated histone H2AX (γ-H2AX), which plays a key role in assembling DNA damage response and repair proteins at the damage sites and provides a rapid, sensitive way to detect the DNA damage event. Our results showed that γ-H2AX+ hepatocytes are increased by albNScko as early as 1 week of age (Fig. 3A). This event peaks at 2 weeks and gradually declines afterward, coinciding with the temporal pattern of Cre expression. TUNEL assays showed that the increase of cell death occurs after the DNA damage event and peaks at 3 weeks (Fig. 3B). Compared to age-matched NSflx/flx mice, 2-week-old albNScko mice showed a modest, but significant, increase in their serum levels of aspartate aminotransferase (AST) and total bilirubin (Fig. 3C). Levels of conjugated bilirubin were undetectable in both albNScko and NSflx/flx mice. These findings are consistent with liver parenchymal damage and not cholestasis at this early age. At 2-3 weeks of age, small nodules appeared in parenchyma of albNScko livers. These nodules contained hepatocytes with more basophilic cytoplasm, NS-positive expression, more BrdU- and Ki67-labeled cells, stronger AFP signals, and less periodic acid Schiff (PAS) staining, compared to hepatocytes outside the nodules (Fig. 3D1). At 2 weeks of age, the regenerative nodules of albNScko livers showed a higher mitotic (Ki67+) activity, compared to NSflx/flx livers of the same age, whereas the perinodular regions showed a much lower mitotic activity (Supporting Fig. 3A). These results, in conjunction with the lack of A6, Sox9, and CK19 expression in the majority of nodular cells (Supporting Fig. 3B), indicate that these nodules contain regenerating hepatocytes, but not bipotential or ductal-like progenitor cells. In contrast to the nonregenerative hepatocytes outside the nodules that contain a single large nucleolus, these newly regenerated hepatocytes contained multiple small nucleoli (Fig. 3D2). Many regenerative nodules were found in close proximity to the hyperplastic bile ductules, such as is shown in the H&E and AFP panels of Fig. 3D1. To determine the spatial contiguity between the regenerative nodules and periportal areas, we performed serial sections to quantify the number of nodules that come in contact with the periportal areas versus those that do not. Of the 19 nodules traced at the age of 2-3 weeks, 16 were directly connected to the periportal region. The three that showed no connection to the ductal region extended beyond the sections collected. Immunostaining showed that the junctional regions between the nodules and periportal areas contained periportal and rare single Sox9+ cells, but not A6+ cells (Supporting Fig. 3C). When albNScko mice grew older than 4 weeks, these discrete nodules became inconspicuous. When albNScko mice reached 12 months of age, surviving hepatocytes in their livers displayed pleomorphic nuclear and nucleolar morphology (Fig. 3E). At this age, NSflx/flx livers show scattered NS signals in a few hepatocytes, but not in CK19-labeled BECs (Fig. 3F1). In contrast, albNScko livers contain regions of mostly NS-low/negative hepatocytes (Fig. 3F2, left upper panel) and restricted areas of strong NS-positive hepatocytes intermixed with NS-low/negative cells (Fig. 3F2, bottom panel). BECs in albNScko livers still show NS-positive signals.
AlbNScko Livers Show Signs of HPSC Activation
The combination of regenerative nodules and BDH suggests that HSPCs may be activated in albNScko livers. Quantitative RT-PCR (qRT-PCR) assays showed that the transcript levels of several HSPC-related genes, including epithelial cell adhesion molecule (EpCAM), CK19, AFP, CD133, CD24, and CK7, are all significantly increased in albNScko livers compared to age-matched NSflx/flx livers (Fig. 4A). The increase of HSPC-related markers is more prominent at 4 weeks of age than at 1 year. Histologically, A6-positive cells appear as early as 3-4 weeks of age in parenchyma and the hyperplastic ductal region of albNScko livers, whereas no A6+ signals are detected in NSflx/flx livers (Fig. 4B). The majority of A6+ cells also coexpress the CK19 antigen on serial sections of 4 um in thickness (Fig. 4C).
CCl4 Increases Hydropic Degeneration and DNA Damage in NS-Depleted Hepatocytes and Stimulates Proliferation of A6+ Cells and Bile Ductules in albNScko Livers
To determine the role of NS in liver regeneration, we analyzed responses of albNScko and NSflx/flx livers to CCl4 treatment at 2 weeks of age when Cre expression and DNA damage were maximal and histological changes and serum measurement of hepatocellular injury were mild in albNScko livers. Liver samples were collected in pairs from CCl4- and oil-treated mice at the first, second, or fourth day of the injection. NSflx/flx livers show acute pericentral necrosis with infiltrating leukocytes during the first 2 days after injection (Fig. 5A1, white arrows). Without CCl4 exposure, albNScko livers contained regenerative nodules and nonregenerative regions (Fig. 5A2, perinodule). In response to CCl4 treatment, albNScko livers began to show not only the same acute pericentral necrosis and leukocyte infiltration as observed in NSflx/flx livers, but also severe hydropic degeneration (black arrows) in the perinodular areas (Fig. 5A3). Notably, the regenerative nodules were relatively resistant to the acute necrotic effect of CCl4 treatment (Fig. 5A4), which is consistent with their lower expression of the key enzyme, CYP2E1, that metabolizes CCl4 and forms free radicals (Supporting Fig. 4A). Mice recovered from CCl4-triggered pericentral necrosis after 4 days (Supporting Fig. 4B). Unlike NSflx/flx livers, which show no increase of CK19+ cells after CCl4-induced damage, albNScko livers displayed a significant increase of CK19+ bile ductules and small CK19+ progenitor-like cells located in the periportal region in response to CCl4 treatment (Fig. 5B and Supporting Fig. 4C). Immunostaining on serial sections showed that the numbers of A6- and CK19 double-positive progenitor cells were increased by CCl4 treatment in albNScko livers (Fig. 5C). Consistently, the number of Sox9 and CK19 double-positive cells was also increased in CCl4-treated albNScko livers (Supporting Fig. 4D). Supporting the idea that HSPCs may be expanded in CCl4-treated albNScko livers, qRT-PCR assays demonstrated that the messenger RNA levels of two HSPC-related markers, EpCAM and AFP, were both up-regulated in albNScko livers after CCl4 treatment (Fig. 5D). The increase of EpCAM occurred within 1 day, peaked on the second day, and dropped after 4 days, whereas the increase of AFP was found primarily on the second day of the injection. Based on the same qRT-PCR assay, we did not detect a significant increase of EpCAM or AFP in NSflx/flx livers during the time window of 1-4 days after CCl4 treatment, which is consistent with the idea that a single dose of CCl4 activates only a small subset of periportal progenitor cells or none.[19, 20]
We then asked whether NS-depleted hepatocytes in the perinodular area are more susceptible to the mitotic stress caused by CCl4-induced damage than are the NS+ hepatocytes in the regenerative nodule. To address this question, we first measured the increase of Ki67+ cells in response to CCl4 treatment in different regions of albNScko livers. In oil-treated albNScko livers, most mitotic (Ki67+) cells were found in the regenerative nodules (25%) and the bile duct epithelium (BDE; 18%), and only a small percentage of the non-regenerative hepatocytes were Ki67+ (1.4%; Fig. 5E). After CCl4 treatment, the number of mitotic cells was increased most significantly on the second day postinjection in perinodular hepatocytes (1.4%-3.2%), regenerative hepatocytes (23.3%-42.3%), and BDE (17.4%-25.2%; Fig. 5E and Supporting Fig. 4E). Next, we examined whether CCl4-induced regeneration may sensitize NS-depleted cells to DNA damage. CCl4 treatment itself did not elicit any DNA damage in NSflx/flx livers (Fig. 5F). In oil-treated albNScko livers, most γ-H2AX+ cells were found in the perinodular areas. Notably, CCl4 increased the percentage of γ-H2AX+ cells in the NS-depleted areas, but not in the regenerative nodules or the BDE (Fig. 5F).
AlbNScko Livers Show a Significant Increase of DNA Damage in Response to PHx
To support the CCl4 results, we performed PHx on albNScko and NSflx/flx mice at 4 weeks of age. At this age, the structure of regenerative nodules in albNScko livers became inconspicuous, and NS-positive and negative hepatocytes were intermixed throughout most of the liver parenchyma. In response to PHx, NSflx/flx livers showed a significant increase of Ki67+ cells that peaked on the second day and recovered mostly on the fourth day. Before the operation, albNScko livers contained more Ki67+ cells than NSflx/flx livers as a result of NSKO-induced liver damage and regeneration. After PHx, albNScko livers showed a blunted and prolonged regenerative response, compared to NSflx/flx livers (Fig. 6A). Though PHx increases Ki67+ cells, but not γ-H2AX+ cells, in NSflx/flx livers, it triggers a significant increase of γ-H2AX+ cells in albNScko livers that continues to rise 4 days after PHx in parallel with the increase of Ki67+ cells (Fig. 6B). The results of CCl4 and PHx experiments both demonstrate that NS deletion predisposes regenerating hepatocytes to DNA damage.
Loss of NS Triggers Replication-Dependent DNA Damage and Perturbs RAD51 Recruitment to HU-Induced Foci in Proliferating Hepatocytes
Primary hepatocytes were isolated from 2-week-old NSflx/flx livers to determine how loss of NS predisposes developing hepatocytes to DNA damage. After two passages, cultured hepatocytes were treated with NS-specific (siNS) and control (siScr) RNA interference duplexes. In the absence of external genotoxic stress, NSKD by siNS significantly increased the percentage of γ-H2AX+ cells (Fig. 7A), ataxia telangiectasia and rad3 related protein (ATR)-positive cells (Fig. 7B, gray bars), and replication protein A-32 (RPA32)-positive cells (Fig. 7B, black bars). A major cause of spontaneous DNA damage is replication stalling, which triggers S-phase DNA damage. To address whether loss of NS predisposes proliferative hepatocytes to replication-dependent DNA damage, we measured the cell-cycle relationship of NSKD-induced DNA damage and showed that NSKD caused a higher percentage of γ-H2AX+ cells in S-phase cells than in non-S-phase cells (Fig. 7C). This DNA-damage profile resembled the effect of HU, a model agent that triggers replication stalling and DNA damage. In support, the DNA damage effect of NSKD is greatly diminished in slowly dividing hepatocytes grown under the serum deprivation condition (Fig. 7D, left panel). This lack of response to NSKD under the low serum condition is not the result of a decrease of NSKD efficiency (Fig. 7D, right panel). In further support, overexpression of NS or its nucleoplasmic mutant, NSdB, both have the ability to protect proliferative hepatocytes from HU-induced DNA damage (Fig. 7E).
To establish that NS is directly engaged in the DNA damage pathway, we first demonstrated that, after HU treatment, the endogenous NS protein in the hepatocytes forms foci in the nucleoplasm without losing its nucleolar signals, and some foci are colocalized with the γ-H2AX+ signal (Fig. 7F). To exclude the possibility that the DNA damage effect of NSKD may be caused secondarily by dys-regulated ribosome biosynthesis, we measured the DNA damage event and the expression levels of pre-rRNAs (ribosomal RNAs) and rRNAs in control-KD and NSKD Hep3B cells in parallel. Pre-rRNA and rRNA species were quantified by qRT-PCR on the processing site (PS)-1, PS-2, PS-3, and 18S rRNA sequences (Supporting Fig. 5A, left diagrams). The different PS-containing products represent precursor species that exist before the processing events occurring at different stages of pre-rRNA processing. Though NSKD reduces NS transcripts and elicits a clear DNA damage response (Supporting Fig. 5B), it has no effect on the processing events occurring on PS-1, PS-2, or PS-3 (Supporting Fig. 5A, right) and neither does it reduce the amount of 18S sequence. Homologous recombination (HR) is the key repair mechanism for replication-induced DNA damage, and knockout of its core protein, RAD51, produces the same early embryonic lethal phenotype as does NSKO. Therefore, we reasoned that NS may play a role in regulating RAD51 recruitment to HU-induced DNA damage foci. To address this possibility, control KD and NSKD hepatocytes were treated with HU (2 mM) for 24 hours and assayed for their RAD51 recruitment efficiency. In control KD cells, HU treatment significantly increased the percentages of γ-H2AX+ cells (30.7%) and RAD51+ cells (38.7%) over non-HU-treated controls (2.8% and 7.0%, respectively; Fig. 7G). In NS-depleted hepatocytes, HU increased γ-H2AX+ cells significantly (37.5%), but its effect on triggering RAD51+ foci was greatly diminished (21.4%; P < 0.01). A direct link between NSKD-induced DNA damage and perturbed RAD51 recruitment was established by the results that overexpression of RAD51 (green fluorescent protein [GFP] tagged), but not that of the GFP control, was capable of partially rescuing the DNA damage phenotype of NS-depleted primary hepatocytes (Fig. 7H). These results indicate that NS depletion predisposes proliferating hepatocytes to replication-dependent DNA damage by perturbing RAD51 recruitment to DNA damage foci.
AlbNScko Triggers DNA Damage and Liver Regeneration
The importance of NS in liver development is shown by the increase of spontaneous DNA damage, apoptosis, BDH, and fibrosis in albNScko livers. DNA damage appears first in albNScko livers during the first to second postnatal week, followed by an increase of apoptotic cells that peaks at 3 weeks of age and the appearance of necrotic foci and regenerative hepatic nodules. Complete loss of NS proteins by albNScko occurs within the first week after birth and mainly affects developing hepatocytes. Although we cannot exclude the possibility that the Alb-Cre transgene is expressed in subsets of BECs, our data indicate that most BECs do not show Alb-Cre activity. This may explain why biliary hyperplasia becomes a prominent feature in adult albNScko livers. Newly generated hepatocytes in albNScko livers form small nodules and display basophilic cytoplasm and multiple small nucleoli. These cells also show higher mitotic activity and NS-positive expression and are less developmentally mature (as evidenced by their AFP-positive and PAS-negative staining), compared to nonregenerative hepatocytes outside the nodule. The close spatial association between the regenerative nodules and periportal areas suggests that newly generated hepatocytes may be derived from non-NS-deficient BECs or HSPCs. In support of this, albNScko livers display increased HSPC-related proteins and the expansion of A6 and CK19 double-positive cells. These findings suggest that HSPCs may be activated by albNScko-induced liver damage. To date, only a handful of mouse genetic models exhibit the phenotype of robust HSPC activation.[22-25] Compared to those published, the albNScko model has the unique features of an early-onset expansion of HSPCs (within 4 weeks of age) and long-term survival (over 1 year).
The role of NS in liver regeneration is shown by the increased NS expression and the response of albNScko livers to CCl4 and PHx. In addition to the phenotypes of acute pericentral necrosis and leukocyte infiltration observed in NSflx/flx livers, CCl4 triggers severe hydropic degeneration in NS-deleted nonregenerative hepatocytes. In contrast, hepatocytes within the regenerative nodules are relatively resistant to the acute necrosis caused by CCl4, which may be explained by their less-differentiated features and lower expression of CYP2E1. Subsequent to CCl4-induced damage, mitotic cells are increased in the BDE, regenerative nodules, and nonregenerative hepatocytes of albNScko livers. Although CCl4 does not induce DNA damage in NSflx/flx livers, it increases DNA damage foci in albNScko livers, but only in the NS-depleted nonregenerative (perinodular) hepatocytes and not in the NS+ regenerative nodule or BDE. Likewise, albNScko livers show increased DNA damage in parallel with a blunted and prolonged regenerative response after PHx. These findings support a role of NS in protecting the genome integrity of regenerating hepatocytes.
Adaptation of albNScko Livers to NSKO-Induced Damage
Despite their early-onset liver pathology, albNScko mice survive more than 1 year. Consistent with their Cre expression level, the DNA damage and cell-death events of albNScko mice subside after 8 weeks, and their HSPC-related protein levels decrease over time as well. We propose that the transient DNA damage effect by albNScko that occurs between the first and eighth week may be the combined result of the Alb-driven Cre expression from birth to 4 weeks of age and the diminishing requirement for NS in hepatocytes as they become more mature and less mitotic. Some newly generated hepatocytes may survive the progenitor stage with a single NS allele and undergo complete knockout only after they become postmitotic. Others may adapt to the NSKO event by silencing the promoter activity of the Alb-Cre transgene or by adopting a semiundifferentiated fate, as reported in Alb-Cre-driven β-catenin knockout mice,[26-29] thereby maintaining their NS expression in old-age livers (Fig. 3F2). Finally, how those newly generated hepatocytes differ from normal mature hepatocytes in their lifespan and metabolic function remains unclear. As aged albNScko livers display a continuous elevation of HSPC-related proteins, and hence a sign of continuous regeneration, we speculate that the lifespan of surviving albNScko hepatocytes may be compromised.
Loss of NS Predisposes Proliferative Hepatocytes to Replication-Induced DNA Damage and Perturbs the Recruitment of RAD51
The DNA damage effect caused by NS depletion is closely linked to the DNA replication event. First, NSKD causes more DNA damage in S-phase hepatocytes than non-S-phase hepatocytes. This DNA damage profile resembles that of HU treatment. Second, NSKD has little DNA damage effect on slowly dividing hepatocytes grown under the low serum condition. Third, overexpression of NS can protect proliferative hepatocytes from DNA damage caused by HU-induced replication stalling. Our data also indicate that NS directly takes part in the DNA damage response/repair pathway based on the reasons that NSKD-induced DNA damage occurs without ribosomal perturbation and that NS protein is recruited to HU-induced nucleoplasmic foci. Importantly, we show that loss of NS does not act by increasing the source of DNA damage, but by perturbing the recruitment of RAD51 to DNA damage foci that occur spontaneously, and that overexpression of RAD51 can functionally rescue the DNA damage effect of NSKD in proliferating hepatocytes.
In conclusion, this study reveals an essential function of NS in maintaining the genome integrity of dividing hepatic progenitors and hepatocytes during liver organogenesis and regeneration. Loss of NS triggers replication-dependent DNA damage by a mechanism that perturbs the recruitment of RAD51 to damage-induced foci. Future studies are needed to further elucidate how NS regulates the recruitment and repair activity of RAD51.
The authors thank the Cellular and Molecular Morphology Core of the Texas Medical Center Digestive Diseases Center (NIDDK-P30-DK056338) and Pamela Parsons for help with immunohistochemistry, the Clinical Pathology Laboratory of Texas Children Hospital for liver function tests, and Dr. Juan Marini (BCM) for help with submandibular bleeding.