Control of Creatine Metabolism by HIF Is an Endogenous Mechanism of Barrier Regulation in Colitis

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis

Louise E. Glovera,b,1, Brittelle E. Bowersa,b, Bejan Saeedia,b, Stefan F. Ehrentrauta,b, Eric L. Campbella,b, Amanda J. Baylessa,b, Evgenia Dobrinskikhb, Agnieszka A. Kendricka,b, Caleb J. Kellya,b, Adrianne Burgessa,b, Lauren Millera,b, Douglas J. Kominskya,c, Paul Jedlickad, and Sean P. Colgana,b,2

aMucosal Inﬂammation Program, bDepartment of Medicine, cDepartment of Anesthesiology and Perioperative Medicine, and dDepartment of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045

Edited by Gregg L. Semenza, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 18, 2013 (received for review February 12, 2013)

Mucosal surfaces of the lower gastrointestinal tract are subject to shifts inherent to mucosal inflammatory lesions. Both HIF-1α and frequent, pronounced fluctuations in oxygen tension, particularly HIF-2α areexpressedininflamed mucosa from IBD patients during inflammation. Adaptive responses to hypoxia are orches- (7) and mouse models of colitis (8). Studies of murine IBD have trated largely by the hypoxia-inducible transcription factors (HIFs). revealed that loss of epithelial HIF-1α correlates with more severe As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that clinical symptoms, whereas constitutive activation of HIF-1 and constitute the barrier between the lumen and the underlying im- HIF-2 is protective (8). Despite this, the molecular targets of HIF-1 mune milieu, we sought to define the discrete contribution of and particularly HIF-2 in IECs are not well characterized. To de- – – fi – HIF-1 and HIF-2 transactivation pathways to intestinal epithelial lineate HIF-1 and HIF-2 speci c target loci, we performed ChIP fi chip analysis of chromatin isolated from hypoxic IECs. We iden- cell homeostasis. The present study identi es creatine kinases fi (CKs), key metabolic enzymes for rapid ATP generation via the ti ed a family of genes involved in creatine (Cr) metabolism that phosphocreatine–creatine kinase (PCr/CK) system, as a unique gene are coordinately regulated by HIF-2. Immunolocalization of the cytosolic “muscle-” and “brain-type” creatine kinases (CKM family that is coordinately regulated by HIF. Cytosolic CKs are ex- and CKB, respectively) revealed coupling to the AJC, and CK pressedinaHIF-2–dependent manner in vitro and localize to apical

inhibition abrogated junctional assembly and barrier function. CELL BIOLOGY intestinal epithelial cell adherens junctions, where they are critical Moreover, dietary Cr supplementation ameliorated the patho- for junction assembly and epithelial integrity. Supplementation genic course of murine colitis. Collectively, these data identify with dietary creatine markedly ameliorated both disease severity a unique role for HIF in modulating the epithelial barrier through fl and in ammatory responses in colitis models. Further, enzymes of regulation of the Cr/CK shuttle. the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease pa- Results fi tients. These ndings establish a role for HIF-regulated CK in epi- HIF ChIP-on-Chip Analysis Identifies Genes Involved in Cr Metabolism. thelial homeostasis and reveal a fundamental link between cellular ChIP was performed in human colon carcinoma Caco-2 IECs bioenergetics and mucosal barrier. using HIF-1α– and HIF-2α–specific polyclonal antibodies (Fig. 1A). ChIP-enriched and input DNA were hybridized to a custom epithelial junctions | energy metabolism | actomyosin | IBD microarray comprising a genome-wide set of predicted transcription

ntestinal epithelia function to both facilitate nutrient transport Significance Iand protect against luminal antigens. Selective permeability is mediated by specialized anatomical features, including dynamic Intestinal epithelial barrier dysregulation is a hallmark of in- intercellular junctions. The apical junctional complex (AJC), flammatory bowel diseases (IBDs). A central role for hypoxic comprising the tight junction (TJ) and subjacent adherens junction signaling has been defined in barrier modulation during in- (AJ), is supported by a highly crosslinked cytoskeleton and is the flammation. We demonstrate that genes involved in creatine key determinant of paracellular permeability and barrier function. metabolism, the creatine kinases (CKs), are coordinately regu- Prominent features of the perijunctional cytoskeleton include an lated by hypoxia-inducible transcription factors (HIFs) and that extensive network of F-actin bundles that associate with TJs, and such regulation is critical to barrier function. Inhibition of the CK a dense circumferential ring of actin and myosin contiguous with pathway abrogates apical junction assembly and barrier in- AJs (1). This actomyosin ring readily copurifies with other cyto- tegrity. Dietary creatine supplementation profoundly attenu- skeletal proteins and demonstrates ATP-dependent contractility ates the pathogenic course of mucosal inflammation in mouse ex vivo. Such observations highlight the contractile nature of colitis models. Moreover, we demonstrate altered expression of the apical actin network and the intimate association between mitochondrial and cytosolic CK enzymes in IBD patient tissue. its components (2). These findings highlight the fundamental contribution of crea- Based on their juxtaposition to the anoxic gut lumen, intestinal tine metabolism to intestinal mucosal function, homeostasis, epithelial cells (IECs) function physiologically in a low-oxygen- and disease resolution. tension microenvironment and exhibit a uniquely adaptive oxygen- ation profile. This profile is prodigiously altered in inflammatory Author contributions: L.E.G. and S.P.C. designed research; L.E.G., B.E.B., B.S., S.F.E., E.L.C., bowel disease (IBD) (3). Adaptive transcriptional responses to A.J.B., A.A.K., C.J.K., A.B., L.M., and D.J.K. performed research; E.D. contributed new oxygen deprivation are mediated primarily through the hypoxia- reagents/analytic tools; L.E.G., B.S., E.D., P.J., and S.P.C. analyzed data; and L.E.G. wrote inducible factor (HIF) complex, comprising a constitutive “β” the paper. subunit, and an oxygen-labile “α” component that is regulated in The authors declare no conflict of interest. part by prolyl hydroxylase (PHD) enzymes (4). Despite their con- This article is a PNAS Direct Submission. current expression in many cell types, HIF-1α and HIF-2α play Data deposition: The data reported in this paper have been deposited in the Gene Ex- nonredundant roles (5) that appear to be highly cell specificto pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE43108). facilitate both short- and long-term adaptations to hypoxia (6). 1To whom correspondence should be addressed. E-mail: [email protected]. Barrier dysregulation with unimpeded flux of luminal anti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gens contributes fundamentally to the profound metabolic 1073/pnas.1302840110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1302840110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. HIF-1 and HIF-2 ChIP-on-chip. (A) Accumu- lation of nuclear HIF-1α and HIF-2α in Caco-2 IECs in normoxia (0 h) and hypoxia. (B) Heat maps gener- ated from normalized smoothed ratios of ChIP enrichment (GENE-E), showing common targets (Left) and discrete promoter occupancy (Right). (Inset) Functional annotation clustering of ChIP–chip hits revealed genes involved in Cr metabolism as HIF-2– speciﬁc targets (C) ChIP–chip results were validated by qPCR analysis following ChIP using anti–HIF-1α, anti–HIF-2α, or IgG. Bar graph represents fold enrichment of ChIP DNA compared with input chromatin (n = 3).

start site (TSS) flanking sequences at 50 bp resolution. Consistent loci displaying a threshold of twofold enrichment in hypoxia relative with previous reports (5), positive hits included cohorts enriched for to an HRE-negative region. Importantly, integration of ChIP– both HIF-1 and HIF-2 binding, as well as loci unique for HIF-1 or chip and ChIP–qPCR analyses confirmed robust HIF-1α en- HIF-2 occupancy alone (Fig. 1B). Bioinformatic analyses for de novo richment of established HIF-1 targets (Fig. 1C). motifs (MEME) and transcription factor binding sites (9) identified In the course of this analysis, we identified the CK pathway as the canonical hypoxia response element (HRE) motif 5′- a unique target of HIF-2. Indeed, CKM featured as one of the most RCGTG-3′ as a highly represented consensus sequence. Inde- significantly enriched HIF-2α–specific loci. Further interrogation pendent ChIP–quantitative PCR (qPCR) analyses confirmed of this chip cluster revealed additional genes implicated in Cr enrichment at discrete loci compared with IgG control, with all metabolism, namely CKB, mitochondrial CK (CKMT1), and the

Fig. 2. HIF-2–dependent up-regulation of CK enzymes. (A) qPCR analysis of CK enzyme (CKM, CKB, CKMT1A) and Cr transporter (SLC6A8)expressioninCaco-2

IECs (20% or 1% O2,6h;n = 4). (B) Immunoblot analysis of CK levels in Caco-2 IECs (1% O2). (C) Immunoblot analysis of nuclear (HIF) and whole-cell lysates (CK) from Caco-2 treated with vehicle (DMSO) or PHD inhibitor (AKB; 100 μM) for 24 h. (D) qPCR analysis of CK expression in shCtrl, shHIF-1α,orshHIF-2α Caco-2 (20%

or 1% O2,6h;n = 4) *P < 0.05. (E)ImmunoblotanalysisofCKlevelsinshCtrl,shHIF-1α, and shHIF-2α IECs (20% or 1% O2,24h).(F, Schematic) Proximal promoter of human CKM and CKB; TSS designated +1. T84 cells were nucleofected (24 h) and cultured in 20% or 1% O2 for 24 h. Data represent ratio of promoter-luc:pRen- Δ luc activity (n = 4; *P < 0.05). (G) Caco-2 IECs were cotransfected with pCK-Luc and pcDNA3.1 vector as control (C) or increasing ratios of pcDNA3.1–HIF-1α ODD or Δ pcDNA3.1–HIF-2α ODD (n = 3; *P < 0.0001). (H and I) Luciferase activity in lysates cotransfected with full-length (WT), truncated pCK-Luc (schematic)(n = 3; *P < 0.0001), or pCK-Luc harboring mutations in distal (HRE2) and proximal (HRE1) sites, and HIF plasmids at a 1:5 ratio (n = 4; *P < 0.05 by ANOVA).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302840110 Glover et al. Downloaded by guest on September 29, 2021 major Cr transporter (SLC6A8)(Fig.1B, Table S1). The CK space is AJ-dependent. Given that CKs accumulate at AJs, func- shuttle defines a key metabolic pathway for temporal and spatial tionally support MII ATPase, and represent HIF-2 target genes, energy buffering. CK regulates cellular ATP through the reversible we speculated that apical junctions may be altered in the absence transfer of high-energy phosphate from phospho-Cr (PCr), con- of HIF-2 signaling. Localization of E-cadherin and ZO-1 in shCtrl necting sites of ATP generation with compartmentalized ATP monolayers revealed linear contours of apical lateral membranes utilization (10). ChIP–qPCR quantitation of HIF binding to pro- (Fig. 3B). In contrast, HIF-2α–depleted cells displayed non- moter regions of CKB and CKM revealed specific occupancy of uniform, undulating junctions, consistent with attenuated in- HIF-2 over HIF-1, with increased enrichment in hypoxia (Fig. 1C). tercellular tension and junction formation (15). Transepithelial To investigate correlation of HIF-2 binding to hypoxia-induced electrical resistance (TER) measurements further indicated abro- gene expression, we evaluated expression of Cr metabolic genes by gated barrier function (57% ± 3.5%) of HIF-2α–depleted IECs qPCR. Transcript levels of Cr transporter (SLC6A8)andcytosolic compared with control monolayers (Fig. S3B). Moreover, assess- + (CKM, CKB) CK isozymes were increased in a time-dependent ment of TER development following Ca2 switch assay revealed manner in hypoxia (Fig. 2A). CK protein levels were similarly that barrier formation of HIF-2α knockdown IECs was significantly increased both in hypoxic Caco-2 lysates (Fig. 2B) and in cells attenuated compared with control cells (Fig. 3C). treated with the PHD inhibitor AKB to stabilize HIF-1α and HIF- + 2α protein under normoxic conditions (Fig. 2C). These data in- CK Contributes to Junctional Assembly After Ca2 Switch. To directly dicate that CK is induced in IECs in response to both hypoxia and assess the contribution of CK to AJC assembly, we monitored pharmacologic HIF stabilization, consistent with HIF-mediated the influence of altered CK signaling on TER development fol- + transcription. lowing Ca2 switch. Cr supplementation markedly enhanced barrier recovery (Fig. 3C), whereas inhibition of CK using dinitro- CK Enzymes Are Induced in Hypoxic IECs in a HIF-2–Dependent fluorobenzene (DNFB) (17) impaired recovery in a dose-dependent fi Manner. To determine the speci city of HIF-induced CK expres- manner (Fig. 3D). CK inhibition correlated with a reduction in sion, we used short hairpin RNA (shRNA) knockdown to in- Cr and PCr metabolite levels, and increased IEC Cr/PCr ratio α α dividually deplete HIF-1 and HIF-2 (Fig. S1A). qPCR analyses (Fig. S3A). To evaluate junction assembly dynamics, IECs were demonstrated that CK mRNA levels were significantly induced by 6 h in response to hypoxia in sh control (shCtrl) and HIF-1α knockdown cells, but not in cells with specific depletion of HIF-2α (Fig. 2D; P < 0.05). Moreover, knockdown of HIF-2α selectively

attenuated basal levels and hypoxic induction of CK protein CELL BIOLOGY (Fig. 2E). Analysis of human CKM and CKB TSS-flanking gene sequences revealed two candidate HRE sites in each promoter (Fig. 2F). To complement our ChIP–qPCR analysis (Fig. 1C), 1.4 kb fragments of CK promoter sequences were cloned upstream of a luciferase reporter. T84 IECs were nucleofected with either pCK- or pHRE-Luc reporter constructs, and exposed to 20% or 1% O2 for 24 h. For each construct analyzed, increased promoter activity was observed in hypoxia (Fig. 2F). To distinguish between HIF-1α– and HIF-2α–mediated influences, pCK-Luc was cotransfected into Caco-2 cells with increasing concentrations of oxygen-stable HIF-α expression vectors. Dose-dependent induction of pCK-Luc was observed with HIF-2α, but not HIF-1α, consistent with ChIP–qPCR and shHIF-2α findings (Fig. 2G). Promoter activity was significantly diminished upon deletion of the proximal HRE (HRE1) in both constructs (Fig. 2H). Muta- tional analysis revealed selective repression of HIF-2–regulated promoter activity upon mutation of HRE1 in pCK-Luc (Fig. 2I). Taken together, these findings indicate that HIF-2 specifically binds to proximal HRE sites in the promoter region of CK genes to directly activate their transcription in IECs.

Cytosolic CK Is Localized to Apical AJs. Differentially localized CK isozymes facilitate a high-energy PCr/CK circuit in polarized cells, wherein mitochondria are located distantly from regions of ATP consumption (10). Previous studies deﬁned CKB and mitochondrial CK as distinct terminals of this circuit in IECs (11, 12). Functional coupling between CKB and myosin II (MII) at the circumferential ring is proposed to confer a spatial energetic advantage for myosin ATPase activity. MII isoforms localize Fig. 3. CK contributes to apical junction assembly. (A) Caco-2 cells were to cadherin junctions, where they regulate AJ development and stained for CKB, CKM, and E-cadherin. (Scale bar, 20 μm.) (B) Lateral mem- force generation through diverse mechanisms (13–15). In po- brane boundaries in Caco-2 shCtrl and shHIF-2α cells stained for E-cadherin μ α– larized IEC, both MIIA and cytosolic CKs colocalized with actin and ZO-1. (Scale bar, 10 m.) (C) shCtrl and HIF-2 depleted T84 cells on permeable inserts were treated with 2 mM EDTA for 5 min and switched and E-cadherin at apical junctions (Fig. S2 A and B), and + to HBSS with normal Ca2 (1.8 mM). TER was measured over time; data rep- coimmunoprecipitation analyses revealed enrichment of MII in resent percent recovery over time relative to baseline values (n = 3, P < fractions precipitated by an anti-CK antibody (Fig. S2C). De- 0.0001 by ANOVA). (D) T84 cells preloaded with 10 mM Cr monohydrate + tailed optical section analysis revealed that CKM and CKB are were subjected to Ca2 switch, and TER measured over time (n = 3, P < highly enriched at AJs, with further distribution of CKM throughout 0.0001 by ANOVA). (E) T84 cells were subjected to Ca2+ switch in the pres- the lateral membrane (Fig. 3A). ence of CK inhibitor DNFB. TER was monitored over time (data relative to no + Intercellular actomyosin forces that support mature apical DNFB; n = 3, *P < 0.05). (F) T84 IECs incubated in low Ca2 media (16 h) + junctions are transmitted through AJs associated with actin ﬁla- before switching to normal Ca2 , with or without 10 μM DNFB, were stained ments (16). Moreover, TJ assembly and sealing of the paracellular for E-cadherin, ZO-1, CKM, and CKB. (Scale bar, 10 μm.)

Glover et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Fig. 4. Cr is protective in TNBS-induced colitis. (A) Mice (n = 15 per group) were fed regular or 2% Cr-supplemented chow for 3 wk before EtOH (Veh) or TNBS administration. Change in body weight was determined (*P < 0.05; **P < 0.001). (B) Kaplan–Meier curve showing survival of chow and 2% Cr-fed mice (log- rank statistic = 18.47; P < 0.0001). (C and D) Histologic sections of colons were scored blindly for pathology indices. (Scale bar, 100 μm.) *P < 0.05; **P < 0.01. (E) Colon anatomy 3 d post-TNBS. Arrows indicate loose stool content in chow-fed mice; arrowheads indicate normal fecal pellet formation in 2% Cr-fed colon. (F) Colonic sections immunostained for CKB and E-cadherin. (Scale bar, 50 μm.) (G) Whole colon KC and IL-6 concentrations (*P < 0.05 by ANOVA, n = 5 mice per group). (H) Colonic chemokine mRNA levels (*P < 0.05 by ANOVA; n = 5 mice per group). (I) Colonic leukocyte populations [neutrophils/poly- morphonuclear cells (PMN), monocytes, dendritic cells, eosinophils] assessed by ﬂow cytometric analysis (n = 10–15; *P < 0.05; **P < 0.01 by ANOVA).

+ cultured overnight in low-Ca2 media to permit cellular de- repressed by 29% ± 6.5%. Analysis of Cr and PCr metabolite polarization and protein translocation. As previously characterized levels in epithelial isolates revealed a significant increase in the − − (18), E-cadherin and ZO-1 localized to subapical ring-like Cr/PCr ratio of Hif-1β / IECs (Fig. S4C), consistent with CK + structures upon extended Ca2 depletion (Fig. 3F), whereas CK inhibition in vitro (Fig. S3A). As an important correlative, we labeling was observed throughout the cytoplasm. One hour post- used the dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene + Ca2 repletion, immunolabeling revealed nascent AJs and initia- sulfonic acid (TNBS) colitis models to ascertain the effect of tion of TJ assembly in control cells. Junctional assembly was altered CK metabolism on IEC permeability. Acute DSS resul- − − markedly retarded in cells treated with DNFB. By 24 h, control ted in significantly increased intestinal permeability in Hif-1β / + + monolayers assumed “mature” AJC lateral membrane staining. mice compared with Hif-1β / littermate controls, as determined Strikingly, CK inhibition resulted in an undulating junctional by translocation of FITC–dextran into serum post–oral admin- − − staining pattern reminiscent of HIF-2α–depleted cells (Fig. 3B). istration (Fig. S4D). Similarly, Hif-1β / mice demonstrated in- Previous studies have shown that a basal level of MII phosphory- creased colitic disease activity in response to TNBS, resulting in lation and activity is necessary for apical junction integrity (13, 19). attenuated survival (Fig. S4E), augmented colonic shortening As such, HIF-mediated induction of CK isozymes might temporally (Fig. S4F), and increased serum FITC–dextran compared with + + buffer PCr supply and ATPase activity during periods of energetic Hif-1β / controls (Fig. S4G; 4.4- ± 1.6-fold; P = 0.0225). Fur- stress. To test this hypothesis, we exposed IECs to 1% O2 in the ther, increased inflammatory cytokine expression was deter- + + presence or absence of DNFB. CK inhibition markedly decreased mined in Hif-1β / mice versus controls (Fig. S4H). Collectively, TERs in T84 monolayers exposed to 1% O2 (Fig. S3; P < 0.01), these findings indicate that IEC Hif signaling mediates a pro- indicating that barrier function is compromised under hypoxic tective barrier effect in these IBD models (8). conditions upon inhibition of the CK/PCr shuttle. Cr Supplementation Attenuates the Severity of Murine Colitis. Bar- Loss of HIF Signaling Results in Altered CK/PCr Circuit in IECs and rier maintenance requires an intricate balance between AJC and Potentiates Murine Colitis. To validate the regulation of CK by cytoskeletal rearrangements that facilitate continual IEC turn- HIF signaling in vivo, we evaluated expression of Ckb in IECs over and transepithelial transport, both energy-dependent pro- derived from Hif-1β IEC-specific knockout mice (Fig. S4A; cesses. Reduced ATP levels have been observed in inflamed IBD − − + + Hif-1β / ) compared with WT (Hif-1β / ) littermates. As outlined biopsies (20), and noninflamed tissues from Crohn disease (CD) in Fig. S4B, Ckb expression was repressed (51.5% ± 5.6%) in patients are more sensitive to uncoupling of oxidative phos- − − Hif-1β / IECs, whereas levels of Pgk1,aHIF-1targetgene,were phorylation (21). Dietary Cr supplementation has been shown to

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302840110 Glover et al. Downloaded by guest on September 29, 2021 confer protection in multiple disease models that display bio- (24). The circumferential actomyosin ring mediates selective energetic dysregulation (22, 23). We thus sought to evaluate the barrier function in both health and disease (25), and is a primary influence of Cr supplementation on mucosal inflammatory path- target for molecular remodeling by diverse inflammatory stimuli ogenesis in murine models of IBD. (26). Moreover, actomyosin contraction is central to homeostatic Mice were fed either normal chow or chow supplemented with and pathologic IEC shedding and barrier restitution (27). Epi- 2% Cr (23), and exposed to 3% (wt/vol) DSS in drinking water. thelial function and barrier integrity is further regulated by low Cr-supplemented animals exhibited markedly reduced suscepti- oxygen tension, through HIF-elicited adaptive pathways (28, 29). bility to DSS-induced colitis, demonstrated by attenuated weight Notable for the present work, a recent study highlighted a role loss and colon shortening (Fig. S5 A and B). Histologic exami- for epithelial Hif-2α in mediating proinflammatory epithelial nation of control Cr-fed mouse colons revealed no overt alterations, responses in murine models of chemical and bacterial colitis, in confirmed by blinded scoring (Fig. S5 C and D). DSS-induced co- contrast to the barrier protective program elicited by Hif-1α (30). litis in chow-fed animals resulted in extensive crypt loss, epithelial As epithelial Hif1signaling has also been shown to promote in- damage (Fig. S5E)andinflammatory cell infiltrate, as well as loss flammation in one study using DSS colitis (31), such findings of solid stool (Fig. S5E). Interestingly, Cr supplementation atten- likely reflect the context-dependent nature of these models. In- uated histologic indices and promoted fecal pellet formation. deed, the temporal kinetics of HIF-1α and HIF-2α in IBD appear Compared with 2% Cr-fed mice, chow-fed DSS mice displayed to be different, with HIF-1α stabilized earlier in the disease increased intestinal permeability (Fig. S5F). Consistent with this, course (as early as day 1 in TNBS colitis) (8) and a more prom- serum and colonic levels of proinflammatory mediators were re- inent HIF-2α signal at later time points (by day 3 in DSS colitis) duced in mice fed 2% Cr compared with chow alone (Fig. S6 A (30). Nonetheless, head-to-head comparisons have yet to be done, and B). HPLC analysis revealed increased colonic Cr in both Cr- and the relative contribution of HIF-1 versus HIF-2 to mucosal fed vehicle and DSS-challenged mice compared with chow-fed signaling defines a key question. In the current work, we identify cohorts (Fig. S6C). Interestingly, DSS challenge itself resulted in CK as a unique HIF target, and define a role for the CK energy elevated Cr levels, indicating that Cr metabolism is altered in shuttle in AJC assembly and epithelial homeostasis. acute mucosal inflammation. Initial studies of the IEC CK shuttle demonstrated functional We next extended these findings to the independent TNBS coupling between CKB and myosin in isolated brush borders, colitis model. Within 2 d of TNBS challenge, chow-fed mice specifically at the circumferential actomyosin ring (11, 12). MII is displayed progressive weight loss relative to vehicles (Fig. 4A; the major IEC cytoskeletal motor that converts ATP hydrolysis P < 0.05). Strikingly, Cr supplementation rendered mice re- into mechanical forces, mediating the static tension and contrac- fi fi fractory to TNBS-induced weight loss. To determine whether tility of actin laments. Interestingly, isoform-speci cknockdown CELL BIOLOGY attenuated wasting translates to a survival benefit in Cr-fed mice, revealed differential roles for MIIA and MIIB in coordinating we recorded mortality following TNBS challenge. Cr supple- E-cadherin–based intercellular junction dynamics (14). Our obser- mentation resulted in enhanced survival over the colitic course vations indicate that CKs localize to apical AJs, where they likely (Fig. 4B). Histologic analysis of colon biopsies (Fig. 4C) revealed constitute one terminal of a functional PCr/CK energy circuit that significant protection conferred by Cr on both injury and in- supplies energy to myosin motors. In addition to PCr generation flammatory indices (Fig. 4D), which correlated with fecal pellet via oxidative phosphorylation coupling, a subset of cytosolic CK formation (Fig. 4E). Vehicle IECs displayed apical enrichment has been shown to associate with glycolytic enzymes to facilitate of CKB that colocalized with E-cadherin (Fig. 4F). With TNBS PCr repletion, wherein HIF-1–mediated induction of the glycolytic challenge in chow-fed animals, patchy disruption of epithelial pathway for ATP generation represents a central adaptation for and crypt architecture associated with loss of E-cadherin junc- cell survival (10). Given the importance of junction integrity to tional staining and colocalization with CKB. In contrast, both epithelial homeostasis, apical ATPases associated with the AJC IEC integrity and junctional staining was preserved in Cr-fed mice seem likely candidates for prioritized PCr targeting under con- exposed to TNBS. To ascertain whether epithelial integrity corre- ditions of energetic stress. Coupled with our findings, this posits an lates with abrogated inflammatory stimuli, we profiled inflammatory mediators and lamina propria leukocyte populations (Fig. S7). Cr supplementation reduced colonic expression of proinflammatory chemokines compared with chow-fed mice (Fig. 4 G and H). Moreover, we identified a significant reduction in both dendritic (P < 0.001) and eosinophil (P < 0.01) populations in Cr-treated versus chow-fed TNBS mice (Fig. 4I). Taken together, these findings strongly imply that Cr supplementation exerts a beneficial effect in colitis-associated mucosal inflammation.

Attenuated CK Levels Suggest Altered Cr Signaling in Intestinal Biopsies from IBD Patients. To investigate the relevance of our findings to human disease, we stained archived paraffin-embedded colon biopsy sections from IBD patients with antibodies to CKB and ZO-1. Similar to murine colonic sections, CKB exhibited dif- fuse cytoplasmic staining in non-IBD tissue, with enrichment at apical junctions colocalized with ZO-1 (Fig. 5A). Interestingly, in colon biopsies from IBD patients, CKB displayed preferential junctional staining relative to cytoplasmic compartments (Fig. 5A). As a corollary, we compared expression of CK enzymes in intestinal biopsies from IBD and non-IBD subjects. A significant decrease in transcript levels of CKM, CKB, and CKMT1 was observed in IBD patient tissue compared with non-IBD controls Fig. 5. CK expression is reduced in chronic IBD. (A) Representative image of (Fig. 5B). These observations implicate marked alterations in the fl colon biopsy from a non-IBD control (Upper) and ulcerative colitis (UC) pa- CK/PCr energy circuit in chronic human mucosal in ammation. tient (Lower) immunostained for CKB and apical TJ marker ZO-1. (Scale bar, 20 μm.) (B) Transcript levels of CKM, CKB, and CKMT1A assessed by qPCR in Discussion cDNA derived from non-IBD control (n = 5), CD (n = 21), and UC (n = 17) Δ The cytoskeletal network that supports apical IEC junctions is individuals. Data expressed as relative Ct values, calculated as 2- Ct (Ct target – among the most highly ordered arrays of actin filaments in nature Ct actin) and analyzed by ANOVA.

Glover et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 intriguing link between HIF signaling and a unique role for Cr/ cancer. A number of studies have now identified reduced levels PCr in junctional energetics. This complex metabolic regulation of CKB in colonic tumors (35, 36), whereas studies using dom- underscores the importance of balancing ATP supply and inant negative CKB mutants have defined a correlation with consumption in cells with high and fluctuating energy demands. molecular markers of epithelial-to-mesenchymal transition in co- In the present studies, Cr supplementation was shown to lon cancer cells (37). Taken together, these observations provide provide substantial benefits in murine colitis. This was mainly a compelling argument for Cr supplementation as an adjuvant attributable to enhanced cellular energetics through the PCr/CK therapy to promote epithelial restitution and ameliorate mucosal circuit, but may also be explained in part by Cr-mediated re- inflammation via enhanced cellular energetics. duction of oxidative stress (10). Cr has been shown to possess antioxidant properties (32), whereas mtCK activation was found Materials and Methods to reduce mitochondrial reactive oxygen species (ROS) (33). These properties define an intriguing overlap with HIF-2 tran- Please refer to SI Materials and Methods for detailed descriptions of scriptional programs, as HIF-2 regulates expression of a number the methods. of antioxidant enzymes (34). Furthermore, loss of HIF-2α results in increased ROS and redox imbalance in multiple cellular Animal Studies. Colitis models were induced as described in SI Materials settings. That HIF-mediated induction of CK may represent and Methods. an antioxidant pathway to buffer redox homeostasis in hypoxic – cells certainly warrants further investigation. ChIP Chip. Input and HIF ChIP-DNA were hybridized to a custom micro- Finally, our data demonstrating attenuated expression of CK array designed to cover a genome-wide set of human promoter regions of ≤2 kb (Switchgear Genomics). All microarray data have been de- enzymes in IBD tissue suggest that intestinal Cr metabolism and posited in National Center for Biotechnology Information’sGeneEx- PCr/CK energetics may be compromised in at least a subset of pression Omnibus (GSE43108). IBD patients. This observation is noteworthy on two levels. First, bioenergetic dysregulation resulting from impaired CK shuttling ACKNOWLEDGMENTS. We gratefully acknowledge Frank Gonzalez (National may contribute to the increased barrier permeability character- Cancer Institute) for his kind donation of the Hif-1β flox mice. This work was istic of inflamed mucosae (25). Second, chronic inflammation supportedbyNationalInstitutesofHealthGrantsDK50189,HL60569,and associated with IBD is a major risk factor for colitis-associated DK095491, and by the Crohn’s and Colitis Foundation of America.

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