PGC-1Β Promotes Enterocyte Lifespan and Tumorigenesis in the Intestine

PGC-1β promotes enterocyte lifespan and PNAS PLUS tumorigenesis in the intestine

Elena Bellafantea,1, Annalisa Morganoa, Lorena Salvatorea, Stefania Murzillia, Giuseppe Di Tullioa, Andria D’Orazioa, Dominga Latorreb, Gaetano Villanib,2, and Antonio Moschettac,d,2

aFondazione Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy; bDepartment of Basic Medical Sciences, Neurosciences and Sense Organs, and cDepartment of Interdisciplinary Medicine, University of Bari Aldo Moro, 70121 Bari, Italy; and dNational Cancer Research Center, Istituto Oncologico “Giovanni Paolo II,” 70124 Bari, Italy

Edited by Steven A. Kliewer, University of Texas Southwestern Medical Center, Dallas, TX, and accepted by the Editorial Board September 9, 2014 (received for review August 12, 2014) The mucosa of the small intestine is renewed completely every 3–5d as antioxidant defense. Both PGC-1α and PGC-1β are pref- throughout the entire lifetime by small populations of adult erentially expressed in tissues with high oxidative capacity where stem cells that are believed to reside in the bottom of the crypts they participate, through mitochondrial biogenesis, in the and to migrate and differentiate into all the different populations metabolic response to high energy demand (4), such as cold- of intestinal cells. When the cells reach the apex of the villi and are adapted thermogenesis in brown adipose tissue (5), fiber-type fully differentiated, they undergo cell death and are shed into the switching in striated muscle (6), and fatty acid β oxidation and lumen. Reactive oxygen species (ROS) production is proportional gluconeogenesis in liver during a fasting state (7, 8). The increase to the electron transfer activity of the mitochondrial respiration in mitochondrial biogenesis and activity stimulated by PGC-1 chain. ROS homeostasis is maintained to control cell death and is proteins may cause an increase in the production of ROS. However, finely tuned by an inducible antioxidant program. Here we show α γ β PGC-1 also has been shown to increase the expression of the that peroxisome proliferator-activated receptor- coactivator-1 major mitochondrial antioxidant enzyme superoxide dismutase 2 (PGC-1β) is highly expressed in the intestinal epithelium and pos- (Sod2) (3, 9). Therefore, PGC-1α is able to upgrade aerobic energy sesses dual activity, stimulating mitochondrial biogenesis and metabolism while preserving ROS homeostasis, by simultaneously oxygen consumption while inducing antioxidant enzymes. To study

β promoting ROS formation and detoxification. Recently, it has been MEDICAL SCIENCES the role of PGC-1 gain and loss of function in the gut, we gener- Drosophila α ated both intestinal-specific PGC-1β transgenic and PGC-1β knock- shown in that the PGC-1 homolog spargel is able to out mice. Mice overexpressing PGC-1β present a peculiar intestinal induce mitochondrial function and oxygen consumption, which is morphology with very long villi resulting from increased entero- coupled to the induction of scavenger systems and ROS reduction, cyte lifespan and also demonstrate greater tumor susceptibility, finally leading to increased longevity (10). On the other hand, in the with increased tumor number and size when exposed to carcino- differentiated intestinal epithelium of mice, PGC-1α induces mito- gens. PGC-1β knockout mice are protected from carcinogenesis. chondrial biogenesis and oxygen consumption, but it is not able to We show that PGC-1β triggers mitochondrial respiration while pro- induce the ROS-scavenging apparatus, thus promoting ROS- tecting enterocytes from ROS-driven macromolecule damage and dependent apoptotic cell death (2). consequent apoptosis in both normal and dysplastic mucosa. Therefore, PGC-1β in the gut acts as an adaptive self-point regula- Significance tor, capable of providing a balance between enhanced mitochondrial activity and protection from increased ROS production. The mucosa of the small intestine is renewed completely every 3–5 d during the entire lifetime through the continuous steps nuclear receptors | gene expression | molecular pathology | colon cancer of proliferation, migration, and differentiation of the cells of the mucosa from the crypt site on the bottom to the villus site he intestine represents the interface between the organism on the top of the mucosa. The factors that regulate enterocyte Tand its luminal environment and is constantly challenged by lifespan and aging are of special interest as related to colon mechanical stress, diet-derived toxins and oxidants, and endog- cancer susceptibility. Here, using genetically modified gain- enously generated reactive oxygen species (ROS), which can and loss-of-function models, we present the importance of the induce serious damage to all biological molecules and cell mitochondrial respiration chain and reactive oxygen species structures (1). To preserve cellular integrity and tissue homeo- homeostasis in the gut and identify the protein peroxisome stasis, the intestine possesses self-renewing capacity via the proliferator-activated receptor-γ coactivator-1β as a gene- continuous migration of new enterocytes that undergo differen- expression modulator of enterocyte lifespan in both normal tiation from the crypt to the apical compartment of the villus, and tumoral conditions. where they become competent to apoptosis and are shed into the Author contributions: E.B., G.V., and A. Moschetta designed research; E.B., A. Morgano, lumen. ROS accumulation within intestinal epithelial cells pro- L.S., S.M., G.D.T., A.D., and D.L. performed research; S.M., A.D., D.L., G.V., and A. Moschetta motes apoptotic cell death in the differentiated compartment contributed new reagents/analytic tools; E.B., G.V., and A. Moschetta analyzed data; and (2). The mitochondrial electron transport chain is a major site of E.B., G.V., and A. Moschetta wrote the paper. ROS production in the cells. Under physiological conditions, the The authors declare no conflict of interest. balance between ROS generation and detoxification is controlled This article is a PNAS Direct Submission. S.A.K. is a guest editor invited by the Editorial by a set of cellular enzymes including superoxide dismutase Board. and catalase. Important components of the ROS-scavenging Data deposition: The data reported in this paper have been deposited in the Gene Ex- pathways are linked to mitochondrial oxidative metabolism via pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE61643). γ 1Present address: Division of Women’s Health, Faculty of Life Sciences & Medicine, King’s the peroxisome proliferator-activated receptor- coactivators College London, London WC2R 2LS, United Kingdom 1α and 1β (PGC-1α and PGC-1β), apparently enabling cells to 2To whom correspondence may be addressed. Email: [email protected] or antonio. maintain normal redox status in response to changing oxida- [email protected]. α β tive capacity (3). PGC-1 and PGC-1 are master regulators This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of mitochondrial biogenesis and oxidative metabolism as well 1073/pnas.1415279111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1415279111 PNAS | Published online October 6, 2014 | E4523–E4531 Downloaded by guest on September 26, 2021 PGC-1β is highly similar to PGC-1α, both in amino acid se- antioxidant systems as well as decreased susceptibility to tumors. quence and ability to regulate several metabolic pathways (8, 11). Indeed, tumors may use adaptive mechanisms to keep their ROS Therefore, in the present study we focus on the function of PGC-1β burden within a range that permits their growth and survival. In in the intestinal epithelium, giving special attention to the such contest, PGC-1β acts as a gatekeeper of redox status, allowing effect of this coactivator in enterocyte homeostasis. We first enterocyte survival and, in cancer-promoting conditions, tumor show that PGC-1β is highly expressed in intestinal epithelium progression. with an almost ubiquitous pattern of localization along the entire crypt–villus axis. To study its activation, we generated mice Results overexpressing PGC-1β selectively in the enterocytes. We show PGC-1β Is Highly Expressed in the Intestinal Epithelium and Modulates that in these cells PGC-1β enhances mitochondrial biogenesis Intestinal Morphology. The role and the exact localization of and respiration and induces a parallel increase in antioxidant PGC-1β in the intestine is completely unknown. Thus, we first enzymes, such as Sod2 and glutathione peroxidase 4 (Gpx4), as investigated the expression levels of PGC-1β in the intestine and well as peroxiredoxins. As a result, the intestinal morphology is its exact localization in the crypt–villus axis of the intestinal severely affected, with significant increases in enterocyte lon- mucosa in wild-type mice. We found significant PGC-1β levels in gevity and mucosal villi length. Concomitantly, PGC-1β over- the entire gastrointestinal tract with clearly higher expression in expression leads to a significant increase in tumor number and the colon (Fig. 1A). Notably, PGC-1β is present along the entire size in two distinct models of intestinal carcinogenesis. More- crypt–villus axis, although it seems to be more highly expressed over, to confirm the role of PGC-1β activity in the intestine, we also in the lowest part of this axis, which corresponds to the villus– generated intestinal-specific PGC-1β (iPGC-1β) knockout mice crypt junction, and in the transit-amplifying crypt compartment, that, in line with the evidence from transgenic mice, show re- as shown by immunohistochemistry (Fig. 1B).This localization duced expression of several metabolic pathways and mitochondrial also was confirmed by mRNA and protein analysis on fractions

Fig. 1. PGC-1β expression and intestinal morphology. (A) PGC-1β mRNA expression in the gastrointestinal tracts in wild-type mice was measured by real-time qPCR. Results are expressed as mean ± SEM. BAT, brown adipose tissue; WAT, white adipose tissue. (B) Paraffin-embedded ileum specimens from wild-type mice were immunoassayed with PGC-1β antibody to determine expression and localization of the protein. (Magnification: 100×.) (C) Paraffin-embedded ileum and colon specimens from wild-type mice and iPGC-1β mice were stained with H&E and observed by light microscopy. Representative specimens are shown. (Magnification: 100×.) (D) The difference in the dimension of intestinal epithelium was quantified by analyzing the length of crypt–villus axis in the ileum and crypts in the colon (n = 8 mice per group). For each mouse an average of five fields (magnification: 100×) is taken in consideration. The two different groups (n = 10) were compared performed using a Student t test followed by a Mann–Whitney u test. *P < 0.05 was considered significant. (E) Wild- type and iPGC-1β mice were injected i.p. with BrdU (1 mL/100 g body weight) and were killed 2 h or 72 h after injection. Paraffin-embedded ileum specimens from wild-type mice and iPGC-1β mice were immunoassayed with BrdU antibody (Roche Applied Science) to determine the migration of BrdU-positive enterocytes. (Magnification: 100×.) (F) BrdU staining per field was quantified by Image J software and reported as percentage per field. The wild-type and transgenic groups (n = 6) at different time points were compared using a Student t test followed by a Mann–Whitney u test. *P < 0.05 was considered significant.

E4524 | www.pnas.org/cgi/doi/10.1073/pnas.1415279111 Bellafante et al. Downloaded by guest on September 26, 2021 isolated from the crypt–villus axis of the intestinal epithelium and iPGC-1β mice. The data showed that overexpression of the PNAS PLUS (Fig. S1A). Although PGC-1β is expressed along the entire PGC-1β coactivator can induce a plethora of genes involved in crypt–villus axis, its expression seems to be higher in the fractions several metabolic pathways (Fig. 2A). The majority of target (V2–C1) that correspond to the middle region of the villus down genes whose expression is enhanced 1.3-fold or more by PGC-1β to the crypt (Figs. S1B and S2C). Although PGC-1α is finely encode for proteins that play an active role in oxidative phos- regulated at transcriptional and posttranslational levels in vari- phorylation (Fig. 2A). Other pathways influenced by the over- ous tissues, the overall expression of PGC-1β is constant, thus expression of PGC-1β are the tricarboxylic acid (TCA) cycle, indicating that PGC-1β serves as a rheostat of mitochondrial the nuclear factor erythroid 2 (NF-E2)-related–like 2 (NRF2)- β function. To investigate the role of PGC-1 in the intestinal mediated oxidative stress response, the glycolysis and gluconeo- β epithelium in a gain-of-function fashion, we generated iPGC-1 genesis pathways, and fatty acid, glutathione, and ubiquinone β β mouse model in which human PGC-1 (hPGC-1 ) is overex- metabolism. These results were confirmed by real-time qPCR pressed selectively in the epithelial cells of the intestine. To this analysis of the expression levels of medium-chain acyl-CoA end, we subcloned the hPGC-1β coding sequence downstream of c β β dehydrogenase (Mcad), cytochrome (cytC), ATP -synthase the villin promoter. The highest levels of hPGC-1 mRNA were (ATPβsynt), and mitochondrial transcription factor A (Tfam), found in the jejunum, where it was 90 times higher as compared which were increased 1.4-, 2.3-, 1.5-, and 1.7-fold, respectively, with mouse PGC-1β (m PGC-1β); the lowest expression was found in the colon, where we observed 23 times higher levels of hPGC-1β vs mPGC-1β (Fig. S1C). These mice express the hPGC-1β transgene along the entire crypt–villus axis as demonstrated by mRNA and protein analysis on different fractions collected from the ilea of transgenic mice (Figs. S1D and S2C). The mRNA analysis shows that the expression of the human coactivator is driven by villin promoter in all the fractions considered but is higher in the fraction corresponding to the most differentiated enterocytes. However, immunohistochemical analysis for PGC-1β protein in ileum specimens from wild-type and iPGC-1β mice reveals an overexpression of the coactivator through the entire axis of transgenic mice, without showing significant differences along the axis (Figs. S1E and S2C). MEDICAL SCIENCES The overexpression of hPGC-1β protein in the intestinal epithelium leads to a striking difference in the morphology of the intestine, specifically in the dimensions of villi and crypts in the small and large intestine, respectively. The epithelium is visibly longer in transgenic than in wild-type mice (Fig. 1C), and quantification of the length shows that the epithelium of transgenic mice is 30% longer than that of wild-type mice (Fig. 1D). This significant variation in the length of intestinal epithelium in the small and large intestine theoretically could be explained either by a higher rate of proliferation forcing the migration of the enterocytes from the crypt along the crypt–villus axis or by a lower rate of apoptosis at the apex of the villi. To address this point, we decided to examine the proliferative state of the enterocytes in the intestinal epithelium of wild-type and transgenic mice using bromodeoxyuridine (BrdU). Mice were killed at 2 h and 72 h after intraperitoneal (i.p.) injection of BrdU to examine BrdU-positive cell migration (Fig. 1E). At the first time point (2 h), BrdU-stained cells were found in the stem cell niches of the crypts and in the transit-amplifying compartment in both wild-type and iPGC-1β mice (Fig. 1E). Strikingly, at 72 h, the BrdU-labeled enterocytes had migrated to the tips of the villi in control mice, but in the transgenic mice the labeled cells reached only halfway up the villus, likely because of the greater length of Fig. 2. Intestinal PGC-1β induces genes involved in mitochondrial function. (A) The gene-expression profiles of ileum samples from wild-type and iPGC- the villi. Notably, 72 h after BrdU injection, although most of the β BrdU staining was lost in the wild-type villi because of apoptotic 1 mice were analyzed by microarray analysis. The metabolic pathways dif- ferentially expressed in wild-type and iPGC-1β mice were identified using shedding of mature enterocytes, the iPGC-1β mice retained the F DAVID software available on the DAVID Bioinformatics Resources website majority of the BrdU-positive cells (Fig. 1 ). Thus, the over- (david.abcc.ncifcrf.gov/). The number of genes up-regulated by 1.5 fold in expression of PGC-1β in the enterocytes leads to significant changes the iPGC-1β mice is indicated for each pathway. (B) Mcad, cytC, ATPβsynt, in the length of the intestinal epithelium without affecting enter- and mitochondrial Tfam mRNAs were measured in ileum specimens from ocyte proliferation. Indeed, these results suggest that the cycling wild-type and iPGC-1β mice by real-time qPCR. Wild-type and transgenic mice stem cells of the transgenic crypts proliferate at the same rate as (n = 6) were compared using a Student t test followed by a Mann–Whitney u those of the wild-type crypts, and the longer villi are the result of test. Results are expressed as mean ± SEM; *P < 0.05. (C) Western blot analysis increased enterocyte lifespan arising from the longer migrating time demonstrates increased COXI and porin protein in enterocytes isolated from iPGC-1β mice as compared with enterocytes from wild-type mice. to the apical apoptotic compartment. (D)PGC-1β overexpression determines the increase in both the endogenous β and COX respiratory capacities of intact enterocytes.A-T,ascorbate/ Intestinal PGC-1 Overexpression Induces Genes Involved in TMPD-dependent oxygen consumption; ER, basal endogenous respira- Mitochondrial Function. To study the transcriptional scenario tion; UR, DNP-uncoupled respiration. *P < 0.05. (E) Enterocytes isolated from that PGC-1β eventually activates in this intestinal context, we iPGC-1β mice present higher enzymatic activity of both Complex IV and cit- performed microarray analysis of ileum samples from wild-type rate synthase. *P < 0.05.

Bellafante et al. PNAS | Published online October 6, 2014 | E4525 Downloaded by guest on September 26, 2021 compared with control mice (Fig. 2B). Notably, Mcad and cytC thus investigated whether increased mitochondrial activity induces mRNA were preferentially overexpressed in the differentiated ROS production in freshly isolated enterocytes from wild-type and compartment (Fig. S1F). Also, both cytC and Mcad showed a iPGC-1β mice. A MitoSOX probe showed that mitochondrial re- gradient of expression in the intestinal epithelium, with higher spiratory chain-dependent generation of ROS levels is lower in expression in the apical compartment than in the crypt pop- iPGC-1β mice than in wild-type mice (Fig. 3A). To investigate the ulation (Figs. S1F and S2C), in agreement with the metabolic status of oxidative stress further, we monitored the ROS-driven switch from glycolytic to oxidative metabolism that is proposed damage to biomolecules in the iPGC-1β enterocytes by staining to occur along the crypt–villus axis (12). Protein analysis revealed sections from wild-type and iPGC-1β mice with antibodies against that both porin and the mitochondrial-encoded cytochrome c 8-oxoguanine (8-oxo-dG) and nitrotyrosine (NITT), which are oxidase subunit I (COXI), which are well-known markers of widely used markers of oxidative stress in DNA and proteins, mitochondrial biogenesis and function, were increased in respectively (Fig. 3B) (3). The levels of 8-oxo-dG and NITT were enterocytes isolated from iPGC-1β mice (Fig. 2C). reduced by 40% and 70% (Fig. 3C), respectively, in transgenic vs. To confirm the increased mitochondrial activity further, we control enterocytes. To investigate if the reduction of macromole- measured mitochondrial endogenous respiratory fluxes in freshly cule damage by ROS could result in a delayed apoptosis at the isolated, intact enterocytes. Maximal uncoupled respiratory ca- apex of villi, we performed a TUNEL assay (Fig. 3C). In line with pacities and ascorbate/N,N,N′,N′,-tetramethyl-p-phenylenediamine the 8-oxo-dG and NITT observations, the TUNEL assay showed (TMPD)–dependent oxygen consumption rates through the in vivo that iPGC-1β mice had fewer apoptotic cells in the apical com- antimycin-isolated COX activity were increased in enterocytes from partment than did wild-type mice (Fig. 3C). To test if the pro- iPGC-1β mice (Fig. 2D). Consistently, COX and citrate synthase tective role of PGC-1β could be exerted through the induction of activity, a nuclear-encoded mitochondrial matrix marker TCA-cycle the expression the antioxidant scavenger enzyme, we performed enzyme, also were increased in total lysate of iPGC-1β enterocytes RT-qPCR analysis of the mitochondrial antioxidant enzymes. (Fig. 2E). In summary, PGC-1β overexpression in the intestine is Indeed, in response to PGC-1β overexpression, the intestine able to induce mitochondrial functions and respiration. shows a significant increase in antioxidant defense, including enzymes such as Sod2, Gpx4, peroxiredoxin 5 (Prdx5), peroxir- Intestinal PGC-1β Overexpression Enhances Antioxidant Defense. An edoxin 3 (Prdx3), thioredoxin 2 (Txn2), and sirtuin 3 (Sirt3) (Fig. efficient mitochondrial respiration is essential for life but produces 3D). Sirt3 is not properly an antioxidant enzyme, because it does ROS that in excess can cause cell damage and, finally, death. We not function as a ROS scavenger, but it is a member of the

Fig. 3. Intestinal PGC-1β induces genes involved in antioxidant defense. (A) A mitoSOX assay was performed on enterocytes from wild-type and iPGC-1β mice. The percentage of cells with positive fluorescence cells was normalized with the respective respiratory activities. Results are expressed as mean ± SEM; *P < 0.05. (B) Paraffin-embedded ileum specimens from wild-type mice and iPGC-1β mice were immunoassayed with 8-oxo-dG antibody to determine oxidative stress in the enterocytes and with NITT antibody, a marker of protein damage. The TUNEL assay was performed on paraffin-embedded samples. (Magnifi- cation: 200×.) (C) Quantitative analysis of immunostaining by 8-oxo-dG and NITT was performed with Image J software. For the apoptotic TUNEL assay the number of apoptotic cells per crypt–villus unit is indicated. (D) Sod2, Gpx4, Prdx5, Prdx3, and Sirt3 mRNAs were measured in ileum specimens from wild-type mice and iPGC-1β mice by real-time qPCR. Results are expressed as mean ± SEM; *P < 0.05.

E4526 | www.pnas.org/cgi/doi/10.1073/pnas.1415279111 Bellafante et al. Downloaded by guest on September 26, 2021 sirtuin family, representing the major mitochondrial deacetylase. PNAS PLUS Therefore, it would be involved in ROS detoxification in- directly, by regulating Sod2 activity (13). Interestingly, the antioxidant enzymes analyzed here were induced in the iPGC-1β mouse mucosa along the entire crypt–villus axis with the highest mRNA expression in the differentiated apical enterocytes (Fig. S2A). The increase in Sod2 expression in the iPGC-1β mice also was confirmed by immunohistochemical analysis in the differentiated compartment of the villus as well as in the crypts (Fig. S2B). Western blot analysis confirmed highest overexpression of Sod2 in the most differentiated fractions of iPGC- 1β villi (Fig. S2C). Overall, PGC-1β overexpression in the intestine decreases ROS-driven macromolecule damage, thus leading to a reduction in apoptotic cell death at the apex of the villi. Together with a higher respiratory rate and more efficient mitochondrial activity, this coactivator can induce the whole battery of mitochondrial antioxidant enzymes, thus prolonging enterocyte survival and conferring the peculiar morphology of the epithelium of the iPGC-1β mice compared with wild-type mice.

Intestinal PGC-1β Overexpression Promotes Intestinal Carcinogenesis. ROS are mutagenic and may stimulate tumorigenesis through the oxidation of DNA and the subsequent accumulation of mutations in key genes involved in cell cycle and proliferation as well as in carcinogenesis. Recently, however, it was demonstrated that activating a ROS-detoxification program promotes tumorigenesis (14). To investigate if the effect of PGC-1β on enterocyte survival also might affect the ROS antioxidant response and the life span in transformed enterocytes, we tested the effects of PGC-1β overexpression MEDICAL SCIENCES Fig. 4. Intestinal PGC-1β promotes chemically and genetically induced in- in two different models of intestinal tumorigenesis. The first testinal carcinogenesis. (A) Gross morphology of colon samples from wild- chemical model consisted of a single i.p. injection with azoxy- type and iPGC-1β mice. (B) Paraffin-embedded ileum and colon specimens methane (AOM) a DNA-alkylating agent facilitating base mis- from wild-type mice and iPGC-1β mice were stained with H&E and observed pairings (15), followed by three cycles of oral dextran sodium sulfate by light microscopy. (Magnification: 25×.) (C) Number of tumors (Left)and (DSS) to sustain intestinal tumor progression via induction of colitis number of tumors categorized by size (Right) per mouse from colons of wild- (16). The second, a genetic model, was generated by crossing type and iPGC-1β mice at the end of the AOM-DSS treatment. The com- min/+ Apc mice (17) with iPGC-1β mice. In the chemical model, gross parison of wild-type and transgenic mice (n = 10) was performed using a – morphology (Fig. 4A) and histological analysis of the intestine Student t test followed by a Mann Whitney u test. Results are expressed as mean ± SEM; *P < 0.05. (D) Intestinal PGC-1β promotes genetically induced revealed worse dysplasia of the tumors in iPGC-1β mice (Fig. 4B), + intestinal carcinogenesis. Surviving 7-mo-old iPGC-1β/Apcmin/ mice pre- + in which both the average number of tumors and average tumor size sented more tumors in the ileum than FVBN/Apcmin/ mice. Tumors in in both C + were increased (Fig. 4 ), as compared with their littermate controls. the ileum and colon were larger in iPGC-1β/Apcmin/ mice than in FVBN/ In the genetic model of intestinal tumorigenesis, the number and Apcmin/+ mice. The comparison of FVBN/Apcmin/+ (n = 19) and iPGC-1β/Apcmin/+ size of the tumors were quantified in 7-mo-old mice, because mice (n = 12) was performed using a Student t test followed by a Mann– 7 mo is the age of onset of tumor formation in the wild-type mice Whitney u test. Results are expressed as mean ± SEM; *P < 0.05. min/+ (FVB/N) crossed with Apc mice (C57BL/6J strain). Notably, in min/+ a group of 20 iPGC-1β/Apc mice, approximately 40% died before reaching the age of 7 mo, whereas there was no mortality at Jun, and Wnt5a were overexpressed, and, conversely, proa- this age in the control littermates. Consistently, the surviving iPGC- poptotic genes Bad, Bid, Fas, Apaf1, and Casp8 were reduced at min/+ 1β/Apc mice showed a significant increase in the number and transcriptional levels. Furthermore, microarray analysis high- min/+ size of tumors in the ileum compared with FVBN/Apc mice lighted potential activated transcription factors (TFs), among (Fig. 4D). Surprisingly, the total number of colon tumors did not which were estrogen receptor-related α (Errα)andthetwoPGC-1 differ between the two mouse groups. However, the size of the coactivators, because of their similar transcriptional activity, and min/+ colon tumors was significantly greater in iPGC-1β/Apc mice TFs involved in cell-cycle and proliferation, such as β-catenin and min/+ than in their age-matched control (FVBN/Apc )(Fig.4D). c-myc (Fig. S4B). However, c-myc levels were similar in the two min/+ Interestingly, the tumors from iPGC-1β/Apc mice main- mouse groups, but cyclin D1 mRNA levels were decreased in tain the overexpression of the PGC-1β transgene (Fig. 5B)as tumors from iPGC-1β mice as compared with tumors from wild- shown in the immunohistochemistry, confirming that the villin type mice, suggesting that the observed phenotype is the result not promoter also is able to drive the expression of the hPGC-1β of further activation of the β-catenin/Tcf4 complex but rather of a during tumor development. In accordance, microarray analysis putative metabolic advantage that PGC-1β overexpression confers performed on tumor specimens from wild-type and iPGC-1β to neoplastic cells (Fig. 5A and Fig. S3A). In this respect, although min/+ mice revealed that the overexpression of the coactivator still is tumors from iPGC-1β/Apc mice showed a significant increase able to induce expression of genes involved in oxidative phos- in mitochondrial and oxidative phosphorylation genes, the antioxi- phorylation, the TCA cycle, glycolysis, and fatty acid metabolism dant enzymes, such as Sod2, as confirmed by histochemical (Fig. S3A). Nevertheless, microarray analysis showed that analysis (Fig. 5B), and Gpx4, Prdx5, Prdx3, Txn2, and Sirt3 were several pathways involved in tumorigenesis, such as ephrin re- increased as compared with tumors from wild-type mice (Fig. 5A). ceptor, mTor, and ERK/MAPK signaling, are up-regulated in To verify that the increased tumorigenesis in mice is further tumors of iPGC-1β mice (Fig. S3A). Interestingly, several genes sustained by the action of antioxidant enzymes that promote involved in molecular mechanisms of cancer were affected by the longevity of transformed enterocytes, we analyzed 8-oxo-dG overexpression of PGC-1β: The protumorigenic proteins Fzd10, and NITT levels by immunohistochemical analysis (Fig. 5C).

Bellafante et al. PNAS | Published online October 6, 2014 | E4527 Downloaded by guest on September 26, 2021 + Fig. 5. Intestinal PGC-1β drives antioxidant enzymes in transformed enterocytes. (A) c-myc levels are similar in tumors from FVBN/Apcmin/ mice and iPGC-1β/ + + Apcmin/ , but iPGC-1β/Apcmin/ mice express higher levels of antioxidant enzymes. cytC, Prdx5, Gpx4, Sod2, Sirt3, Txn2, and Prdx3 mRNAs were measured in ileum specimens from wild-type mice and iPGC-1β mice by real-time qPCR. Results are expressed as mean ± SEM; *P < 0.05. (B and C) Paraffin-embedded tumor specimens from FVBN/Apcmin/+ mice and iPGC-1β/Apcmin/+ mice were immunoassayed with PGC-1β and Sod2 (B) or 8-oxo-dG and NITT (C) antibodies. A TUNEL assay was performed on paraffin-embedded samples. (Magnification: 200×.) (D) Quantitative immunostaining analysis for 8-oxo-dG, NITT, and TUNEL was performed with Image J software. *P < 0.05.

In line with evidence obtained in normal mucosa, PGC-1β over- (ileum) and colon (Fig. S4A). However, expression analysis did expression in tumors provided protection against ROS-driven not succeed in showing complete knockout of PGC-1β because macromolecule damage, as shown by the reduction of 8-oxo-dG of the presence of cells that do not express villin (i.e., muscle and NITT staining in transgenic mice as compared with the cells) in the intestinal tube (Fig. 6A). In line with the effect of control group (Fig. 5 C and D). Furthermore, the TUNEL assay PGC-1β overexpression on the transcription of several metabolic min/+ showed that the tumors of iPGC-1β/Apc mice had 80% pathways, microarray analysis of ileum samples from PGC-1β fl/fl fewer apoptotic cells (Fig. 5 C and D), confirming that PGC-1β is and PGC-1β fl/+ mice (hereafter generically referred to as a master regulator of longevity in transformed cells. In conclu- “PGC-1β fl/? mice”) and iPGC-1βKO mice revealed that PGC-1β sion, PGC-1β overexpression, also sustained by villin promoter deletion in enterocytes led to the down-regulation of numerous during tumorigenesis, is able to promote mitochondrial effi- pathways involved in metabolic cascades, such as oxidative ciency and activate the ROS scavenger systems thus creating phosphorylation (24 genes down-regulated in iPGC-1βKO mice in permissive conditions for tumor progression. comparison with the control group), the TCA cycle, glycolysis, gluconeogenesis, and fatty acid metabolism (Fig. S4B). To verify the Intestinal PGC-1β Ablation Decreases Antioxidant Defense and Intestinal effect of PGC-1β on target genes in the large intestine, we per- Carcinogenesis. To confirm the key role of PGC-1β in intesti- formed real-time qPCR on colon samples from PGC-1β fl/? and nal homeostasis and tumorigenesis, we generated iPGC-1βKO iPGC-1βKO mice for genes implicated in oxidative phosphorylation mice by crossing villinCre transgenic mice that express Cre (cytC), the TCA cycle (isocitrate dehydrogenase 3 subunit α, recombinase under the control of villin promoter with PGC-1β Idh3α), and mitochondrial antioxidant enzymes. We found a 30– flox/flox (PGC-1β fl/fl) mice whose PGC-1β cassettes are flanked 40% decrease for all genes except Prdx5, which showed only a slight by three LoxP sites. To verify the efficiency of Cre recombinase reduction (Fig. 6B). Although the transcriptional activity of PGC-1β expression within enterocytes and its ability to delete PGC-1β in the intestine also was confirmed by its deletion within enter- cassettes, we performed real-time qPCR and immunohisto- ocytes, the morphological effect on intestinal homeostasis observed chemistry for PGC-1β. Indeed, mRNA expression levels of the with its overexpression was not confirmed in the normal mucosa of coactivator were decreased dramatically both in small intestine iPGC-1βKO mice. Indeed, the intestines of knockout mice

E4528 | www.pnas.org/cgi/doi/10.1073/pnas.1415279111 Bellafante et al. Downloaded by guest on September 26, 2021 PNAS PLUS MEDICAL SCIENCES

Fig. 6. Intestinal PGC-1β ablation decreases antioxidant defense and intestinal carcinogenesis. (A) Paraffin-embedded ileum and colon specimens from PGC- 1β fl/? and iPGC-1βKO mice were immunoassayed with PGC-1β antibody to verify its deletion within enterocytes. (Magnification: 100×.) (B) CytC, ATPβsynt, Idh3a, Sirt3, Sod2, Txn2, Prdx3, and Prdx5 mRNAs were measured in colon specimens from PGC-1β fl/? and iPGC-1βKO mice by real-time qPCR. The comparison of wild-type and transgenic mice (n = 6) was performed using a Student t test followed by a Mann–Whitney u test. Results are expressed as mean ± SEM; *P < 0.05. (C) Gross morphology of colon samples from PGC-1β fl/? and iPGC-1βKO mice. (D) Paraffin-embedded ileum and colon specimens from PGC-1β fl/? and iPGC- 1βKO mice were stained with H&E and observed by light microscopy. (Magnification: 25×.) (E) Number of tumors (Left) and number of tumors categorized by size (Right) per mouse from colon of PGC-1β fl/? and iPGC-1βKO mice at the end of the AOM-DSS treatment. The comparison of PGC-1β fl/? and iPGC-1βKO mice (n = 10) was performed using a Student t test followed by a Mann–Whitney u test. Results are expressed as mean ± SEM; *P < 0.05. (F and G) Paraffin- embedded ileum specimens from PGC-1β fl/? and iPGC-1βKO mice were immunoassayed with 8-oxo-dG antibody (F) and with NITT antibody (G). Magnifi- cation: 100×.

appeared to be similar to those of PGC-1β fl/? control mice, oxidative phosphorylation, glycolysis, and the Krebs cycle and and the length of villi and crypts as well as migration along the of mitochondrial antioxidant enzymes affecting oxidative status crypt–villus axis (followed through BrdU injection) did not without altering intestinal homeostasis in physiological conditions. differ in iPGC-1βKO and PGC-1β fl/? mice (Fig. S4C). However, Conversely, the absence of PGC-1β protected mice against chemi- when we challenged PGC-1β fl/? and iPGC-1βKO mice with the cally induced tumorigenesis. AOM/DSS protocol, PGC-1β ablation protected the intestine against tumorigenesis. Indeed, gross morphology of iPGC-1βKO Transcriptional Regulatory Network of PGC-1β. PGC-1β can interact colon samples showed fewer tumors than in PGC-1β fl/? mice with several members of the nuclear receptor family as well as (Fig. 6C). Histological analysis also revealed worse dysplasia (Fig. several non-nuclear receptor TFs to respond to diverse stimuli and 6D), with a greater number and size of tumors in PGC-1β fl/? regulate different pathways. To identify the TFs coregulated by this mice than in knockout mice (Fig. 6E). In contrast with transgenic coactivator within the enterocytes, we sought to use next-genera- mice, in which, despite increased mitochondrial activity, the an- tion DNA sequencing to determine the genome-wide binding sites tioxidant machinery was able to cope with ROS-driven damage to for PGC-1β. We performed ChIP with a PGC-1β antibody in macromolecules, the normal and dysplastic mucosa of iPGC- enterocytes isolated from wild-type, iPGC-1β,PGC-1βfl/?, and 1βKO mice were more susceptible to oxidative stress, as dem- iPGC-1βKO mice. PGC-1β–associated DNA was isolated by im- onstrated by increased 8-oxo-dG and NITT staining in knockout munoprecipitation, processed, and subjected to high-throughput as compared with control mice (Fig. 6 F and G). In summary, DNA sequencing using the Illumina Genome Analyzer HiSEq intestinal-specific ablation of PGC-1β led to decreased expression 1000 platform. As control, input DNA isolated under the same of target genes involved in several metabolic pathways, including procedure was sequenced. Genomic locations were obtained for

Bellafante et al. PNAS | Published online October 6, 2014 | E4529 Downloaded by guest on September 26, 2021 peaks in the University of California, Santa Cruz (UCSC) RefSeq track of the Mouse NCBI37/mm9 assembly. The analysis of the underlying identified PGC-1β peaks through Genomatix MatInspector software revealed known and previously un- identified transcriptional partners of PGC-1β on the basis of the enrichment of their consensus DNA-binding motifs. Interestingly, motif analyses revealed a greater enrichment of several TFs involved in metabolism and intestinal homeostasis, such as PPARs, forkhead box protein O1 (Foxo1), Mef, nuclear respiratory factors (NRFs), and sterol regulatory element-binding protein (Srebp)-binding domains, in iPGC-1β enterocytes than in the control group (Fig. 7A). In accordance, iPGC-1βKO mice showed reduced enrichment of some of these TF-binding sites (Fig. 7A). To study the connection between PGC-1β and these TFs in both gain and loss of function models, we exploited the Ingenuity pathway analysis and the prediction of TF activity based on the up-regulation or down-regulation of target genes in iPGC-1β and iPGC-1βKO ileum specimens, respectively. A Venn diagram was used to classify those TFs. The majority of the overlapping TFs has been observed in the groups of predicted receptors activated/ inhibited between iPGC-1β and iPGC-1βKO mice, including the two PGC-1 coactivators themselves. However, the presence of several TFs shared by three groups (e.g., Srebp1c, Foxo1, Hnf-1, the Ppars, Errα, and Hif-1) suggested that they may play a role in the observed phenotype, given that they are so intrinsically linked with PGC-1β transcriptional activity and function. Most interestingly, NRFs were the only TFs shared by all the examined groups, indicating their potential role in extending enterocyte lifespan and in tumorigenesis via PGC-1β (Fig. 7B). Discussion This work shows that the TF coactivator PGC-1β is abundantly expressed along the entire crypt–villus axis in the intestine and plays an important role in the regulation of enterocyte energy production and lifespan. Indeed, its up-regulation in normal and transformed intestinal cells exerts an antiapoptotic role, both in normal mucosa and in protumorigenic conditions. PGC-1β overexpression in enterocytes can stimulate mitochondrial functions through the induction of key enzymes involved in oxidative phosphorylation, the TCA cycle, and pyruvate and fatty acid metabolism and causes a significant change in the intestinal morphology, with a great increase in the length of the villi in the small intestine and of crypts in the colon. This striking phenotype is the result not of an increased proliferation rate but, instead, of a reduction in apoptosis in the apical compartment of the intestinal epithelium. PGC-1β overexpression renders enterocytes less susceptible to ROS-driven macromolecule damage, thus leading to a delay in apoptotic events in the differentiated compartment. β Indeed, PGC-1β possesses dual activities: It stimulates mitochon- Fig. 7. Transcriptional regulatory network of PGC-1 .(A) Chip-Seq analysis performed with chromatin from enterocytes of wild-type and iPGC-1β mice drial electron transport, and it also is able to induce antioxidant (Upper) and PGC-1βfl/? and iPGC-1βKO mice (Lower) immunoprecipitated enzymes, such as Sod2, Gpx4, and peroxiredoxins. Intriguingly, with PGC-1β antibody. The analysis was performed with biological dupli- overexpression of PGC-1β produces a significant increase in tumor cates; results are shown as the sum of binding sites for each group. (B) Venn growth rate in two distinct models of intestinal carcinogenesis, be- diagram for TFs whose binding site enrichment is higher in iPGC-1β mice cause it induces the expression of antioxidant enzymes, thus leading (group 1) or is reduced in iPGC-1βKO mice (group 2) and TFs that were reduced ROS damage and apoptosis in transformed enterocytes. predicted by Ingenuity pathway analysis to be activated (group 3) or Moreover, the absence of PGC-1β within enterocytes leads to the inhibited (group 4) in transgenic and knockout intestines, respectively. down-regulation of those metabolic pathways that are greatly enhanced by the overexpression of the coactivator in the intestine, suggesting a direct link between PGC-1β and these metabolic cas- mental for life, it is not surprising that mechanisms increasing cades. Furthermore, in line with the protumorigenic phenotype mitochondrial activity are linked so tightly to an anti-ROS genetic β observed in iPGC-1β mice, intestinal PGC-1β deficiency seems program. Thus, PGC-1 seems to act as an adaptive self-point to be protective against tumorigenesis induced by chemical agents. regulator, capable of providing a balance between enhanced mito- The dual action of PGC-1β on the induction of genes involved in chondrial activity and cytotoxic protection from increased ROS mitochondrial functions and antioxidant enzymes provides a clear production. Although high ROS levels are harmful to normal cells, mechanism by which tissues such as skeletal muscles, brown fat, and they have long been thought to aid tumor development in several others can ramp up mitochondrial metabolism to deal with altered ways: by inducing DNA damage and subsequent mutations, by ac- external conditions without causing self-induced oxidative damage. tivating inflammatory pathways, and by stabilizing hypoxia-inducible Because alterations in the rate of oxidative metabolism are funda- factor (18, 19). These cancer-promoting effects of ROS suggest that

E4530 | www.pnas.org/cgi/doi/10.1073/pnas.1415279111 Bellafante et al. Downloaded by guest on September 26, 2021 the use of antioxidant compounds would reduce cancer risk. generated by injecting the transgene plasmid digested with HpaI into the PNAS PLUS However, although many cancer cell types have increased levels of pronuclei of the fertilized eggs of FVB/N mice. Mice carrying the transgene ROS, they must restrict ROS levels and/or adjust the signaling were identified by PCR of genomic DNA to confirm the presence of the pathways that are dependent on cellular redox status below a given hPGC-1β coding sequence. Stomach, liver, brain, kidney, jejunum, duode- threshold so as to escape cell death. Among the potential TFs that num, ileum, and colon of transgenic mice were dissected and prepared for may collaborate with PGC-1β, NRFs seem to be the most reliable total RNA extraction and immunohistochemistry to evaluate the specific of the TFs responsible for the increased enterocyte lifespan and intestinal expression of transgene under the villin promoter control. To tumorigenesis. Indeed, tumors may use adaptive mechanisms to generate iPGC-1βKO mice, we crossed villinCre transgenic mice (Jackson keep their ROS burden within a range that permits growth and Laboratory) with PGC-1β flox/flox (PGC-1β fl/fl) mice. As control group we survival without affecting the mitochondrial competence that is used both PGC-1β fl/fl and PGC-1β fl/+ mice (generically referred to as “PGC-1β ” β Min/+ β important for cancer cell viability (20). In such contest, PGC-1β fl/? mice ). iPGC-1 Apc mice were generated by crossing iPGC-1 + acts as a gatekeeper of redox status, allowing enterocyte survival transgenic mice with C57BL/6-ApcMin/ mice (Jackson Laboratory). All and thus increasing their lifespan and, in cancer-promoting the experiments presented in this study were carried out in male mice. conditions, tumor progression. The experimental protocol was approved by the Ethical Committee of the Consorzio Mario Negri Sud and also was certified by the Italian Ministry Materials and Methods of Health according to internationally accepted guidelines for the animal care. To generate the pSKvillin PGC-1β, first the hPGC-1β (3.1-kb) fragment with XhoI and KpnI restriction sites was generated by PCR from pcDNA3 PGC-1β plasmid (kindly provided by Antonio Vidal-Puig, Department of Clinical ACKNOWLEDGMENTS. We thank Salvatore Modica for support during the study. This work was funded by Italian Association for Cancer Research Biochemistry, University of Cambridge, Cambridge, United Kingdom). Then Grant IG 14732; Italian Ministry of University and Education Fund for the fragment was subcloned at the XhoI and KpnI restriction sites down- Investments in Basic Research Grant IDEAS RBID08C9N7 and Grant PRIN stream of the villin promoter region of the pSKVillin plasmid (kindly pro- 2010FHH32M-002; Italian Ministry of Health Young Researchers Grants vided by Deborah Gumucio, Department of Cell and Developmental Biology, GR-2008-1143546 and GR-2010-2314703; and University of Bari Grant IDEA University of Michigan, Ann Arbor, MI). The iPGC-1β transgenic mice were GRBA0802SJ-2008.

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