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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2015 Nov;185(11):2983–2993. doi: 10.1016/j.ajpath.2015.07.005

Id1 Expression in Endothelial Cells of the Colon Is Required for Normal Response to Injury

Ning Zhang ∗,†, Kotha Subbaramaiah ∗, Rhonda K Yantiss ‡, Xi Kathy Zhou §, Yvette Chin †, Ellen J Scherl ∗, Brian P Bosworth ∗, Robert Benezra †,∗∗, Andrew J Dannenberg ∗,∗
PMCID: PMC4630175  PMID: 26348574

Abstract

Inhibitor of DNA binding (ID)-1 is important for angiogenesis during embryogenesis and tumor development. Whether ID1 expression in endothelial cells of the colon is required for normal response to injury is unknown. We demonstrate that Id1 is up-regulated in colonic endothelial cells in an experimental model of colitis and in the inflamed mucosa of patients with inflammatory bowel disease. Because prostaglandin E2 and tumor necrosis factor-α are also elevated in colitis, we determined whether these factors could induce ID1 transcription in cultured endothelial cells. Tumor necrosis factor-α stimulated ID1 transcription via early growth response 1 protein (Egr-1). By contrast, the induction of ID1 by prostaglandin E2 was mediated by cAMP response element–binding protein (CREB). To determine whether the increased ID1 levels in the endothelial cells of inflamed mucosa were an adaptive response that modulated the severity of tissue injury, Id1 was conditionally depleted in the endothelium of mice, which sensitized the mice to more severe chemical colitis, including more severe diarrhea, bleeding, and histological injury, and shorter colon compared with control mice. Moreover, depletion of Id1 in the vasculature was associated with increased CD31+ aggregates and increased vascular permeability in inflamed mucosa compared with those in Id1 wild-type control mice. These results suggest that endothelial ID1 up-regulation in inflamed colonic mucosa is an adaptive response that modulates the severity of tissue injury.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn disease, is a systemic disease characterized by chronic, relapsing inflammation of the gastrointestinal tract. IBD affects about 5 in 1000 people in North America.1 The precise cause of IBD is unknown but is presumed to result from environmental stimuli, including the microbiome in genetically susceptible hosts.2 The injured mucosa displays variably severe neutrophil infiltration, increased epithelial cell proliferation, and enhanced angiogenesis.3, 4 Levels of tumor necrosis factor (TNF)-α and cyclooxygenase-derived prostaglandin (PG) E2 are increased in IBD and play an important role in both tissue injury and angiogenesis.5, 6, 7, 8, 9, 10, 11 The mechanisms by which these factors affect the mucosa are complex and incompletely understood.

Inhibitors of DNA binding (ID) proteins are key regulatory factors in a wide range of developmental and cellular processes. These proteins inhibit the activity of helix-loop-helix transcription factors, retinoblastoma protein, and members of the E26 transformation-specific protein family.12

There are four members of the ID family, ID1 to 4. Of these, ID1 plays an important role in maintenance of self-renewal and pluripotency in adult stem cell populations, including those of colonic crypts.13, 14, 15 ID1 plays a role in both gastrointestinal neoplasia and colitis.16, 17, 18 Id1 deficiency promoted injury in a mouse model of chemically induced colitis; injury was more severe in animals with globally depleted Id1 expression compared with those with Id1 depletion limited to the colonic epithelium.15 These results suggest that ID1 may be an important regulatory protein in colonic epithelial cells as well as other cell types, such as endothelial cells. Given the importance of ID1 in angiogenesis in various tumor types,19, 20 in this study, we hypothesized that ID1 expressed in endothelial cells is involved in the inflammatory response to colonic injury.

Thus, the goals of the study were to determine whether endothelial cells show increased ID1 expression in the setting of colitis; to evaluate the relationships between PGE2, TNF-α, and ID1 expression in endothelial cells; to and to determine whether selective depletion of Id1 in endothelial cells affects the severity of colitis. Evidence is presented that Id1 is up-regulated in endothelial cells in both experimental colitis and IBD. This finding appears to be an adaptive response that regulates the severity of tissue injury.

Materials and Methods

Materials

Endothelial cell media were purchased from ScienCell Research Laboratories (Carlsbad, CA). TNF-α, α smooth muscle actin, and β-actin antisera, normal mouse IgG, and Evans blue dye were purchased from Sigma-Aldrich (St. Louis, MO). CD31 antiserum was purchased from Dianova (Hamburg, Germany). Lymphatic vessel endothelial hyaluronan receptor (LYVE) 1 antiserum was purchased from R&D Systems (Minneapolis, MN). Hypoxia inducible factor (Hif)-1α antibody was purchased from Novus Biologicals (Littleton, CO). PGE2 was purchased from Cayman Chemical Co. (Ann Arbor, MI). [32P]CTP was purchased from PerkinElmer Life Sciences (Boston, MA). Random priming kits were purchased from Roche Applied Science (Indianapolis, IN). Nitrocellulose membranes were purchased from Schleicher & Schuell (Keene, NH). Reagents for the luciferase assay were purchased from Analytical Luminescence (San Diego, CA). The human β-actin cDNA was purchased from Ambion, Inc. (Austin, TX). Antisera to Id1, phospho cAMP response element–binding (CREB) protein, and early growth response (EGR)-1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Western blot analysis detection reagents (ECL) were purchased from Amersham Biosciences (Piscataway, NJ). Plasmid DNA preparation kits and pSVβgal were purchased from Promega Corp. (Madison, WI). RNeasy mini kits were purchased from Qiagen (Valencia, CA). Oligonucleotides were synthesized by Sigma-Genosys (The Woodlands, TX). Chromatin immunoprecipitation (ChIP) assay kits purchased were from Upstate Biotechnology, Inc. (Lake Placid, NY). siRNAs to CREB and EGR-1 were purchased from Dharmacon Inc. (Lafayette, CO). The expression vector for ID1 was purchased from Open Biosystems, Inc. (Huntsville, AL). Id1 promoter deletion and mutant constructs have been described previously.21, 22

Human Tissue

Thirteen surgical specimens were obtained from patients with medically refractory ulcerative colitis (n = 6) or Crohn disease (n = 7). Representative tissue sections from inflamed areas were selected for analysis and immunohistochemistry (IHC) stains. Five cases of acute diverticulitis were selected for ID1 IHC analysis. Normal, noninflamed tissues from the surgical resection margins of 10 colons served as controls; indications for resection included IBD, a history of diverticulitis, and cancer. The study protocol was approved by the Committee on Human Rights in Research at Weill Cornell Medical College.

Animal Models

Inbred male mice 8 to 10 weeks old were used. Endothelium-specific Id1 knockout (Id1ΔEndo) mice were generated by crossing Id1fl/fl mice14 with Cdh5-CreERT2 transgenic mice,23 which expressed a tamoxifen-inducible form of CreERT2 under the control of VE-cadherin regulatory sequences. Id1−/− mice have been described previously.24 Mice were group-housed under specific pathogen-free conditions and allowed unrestricted access to standard mouse chow. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center.

Induction of Experimental Colitis

Experimental colitis was induced in mice by the administration of 2.5% (w/v) dextran sulfate sodium (DSS) (molecular weight, 36 to 50 kDa; MP Biomedicals, Irvine, CA) in water ad libitum over a 7-day period and then a switch to regular drinking water for 14 days. The severity of diarrhea and bleeding was determined by an investigator (N.Z.) who was blinded to the identity of the mouse genotype. Deletion of Id1 in endothelium was achieved by i.p. injection with 200 μL tamoxifen in corn oil at 10 mg/mL on days 1 and 8.

Histological Assessment of Experimental Colitis

Mouse colons were Swiss-rolled and fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin. The degree of colitis was assessed by a modified scoring system described by Suzuki et al,25 also taking into consideration the extent of the lesion. Briefly, each focus of colonic injury was graded for severity: 1 indicated loss of the basal one-third of the crypts with mild mucosal inflammation and edema; 2 indicated loss of the basal two-thirds of the crypts with moderate mucosal inflammation; 3 indicated retained surface epithelium with complete loss of crypts as well as inflammation and granulation tissue in the mucosa; and 4 indicated ulcerated and inflamed mucosa and submucosa without residual epithelium. These values were added and the means calculated to provide a severity grade. The extent of inflammatory changes in each specimen was also evaluated: 1 indicated unifocal, involving <10% of the colon; 2 indicated multifocal, affecting 10% to 50% of the colon; and 3 indicated extensive injury, affecting >50% of the examined colon. An overall colitis score reflected the sum of the grades of severity and extent. The histological assessment was performed by a gastrointestinal pathologist (R.K.Y.) who was blinded to the genotype of the mice.

IHC Analysis

Five-micron tissue sections were deparaffinized, rehydrated, and incubated in 3% H2O2 for 20 minutes to block endogenous peroxidase activity. After antigen retrieval, slides were blocked with 10% normal goat serum and 2% bovine serum albumin for 30 minutes. Slides were incubated with anti–mouse-specific Id1 rabbit monoclonal antibody (Biocheck, Foster City, CA; 1:100 dilution) and anti–mouse-human Id1 cross-specific rabbit monoclonal antibody (Biocheck; 1:500 dilution) for 2 hours, followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA; 1:200 dilution) for 1 hour.26 A Diaminobenzidine (DAB) Map Kit was used for detection per the manufacturer's instructions (Ventana Medical Systems, Tucson, AZ). Slides were reviewed by a gastrointestinal pathologist (R.K.Y.) who was blinded to the diagnosis (human tissues) and treatment group (mouse studies). IHC analysis was performed with rat anti-mouse CD31, goat anti-mouse LYVE 1, mouse anti-mouse α smooth muscle actin, and rabbit anti-mouse Hif-1α (1 μg/mL each) at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center, using a Discovery XT Processor (Ventana Medical Systems). CD31+ aggregates were scored by an investigator (R.B.) blinded to the genotype of the mice, on a three-point scale: 0 indicated no aggregates; 1 indicated occasional aggregates; and 2 indicated frequent aggregates.

Vascular Permeability

Colonic vascular leakage was determined by quantification of the amount of Evans blue dye that had been extravasated. Evans blue dye (0.5% in saline) was injected into the tail vein 2 hours before tissue harvest. Systemic vasculature was perfused with 5 mL normal saline. Colons were removed and dried at 56°C overnight. The weight of the dried colon was determined. Subsequently, the Evans blue dye was extracted from the colon using formamide at 37°C for 24 hours and quantified using a spectrophotometer at 610 nm.27

Cell Culture

Human umbilical vein endothelial cells and human intestinal microvascular endothelial cells were purchased from ScienCell Research Laboratories. Cells were maintained in endothelial cell culture medium. All cells were grown to 60% confluence in a 5% CO2 water-saturated incubator at 37°C before being placed in serum-free medium for 24 hours. Subsequently, treatments were performed in serum-free medium.

Western Blot Analysis

Cell lysates were prepared by the treatment of cells with lysis buffer [150 mmol/L NaCl, 100 mmol/L Tris (pH 8.0), 1% Tween 20, 50 mmol/L diethyldithiocarbamate, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/mL trypsin inhibitor, and 10 μg/mL leupeptin]. Lysates were prepared by sonication of cells for 20 seconds on ice and centrifugation at 10,000 × g for 10 minutes to sediment the particulate material. The protein concentration of the supernatant was measured as described by Lowry et al.28 SDS-PAGE was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli and colleagues.29 The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al.30 The nitrocellulose membrane was then incubated with primary antibodies. Secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL Western blot detection system according to the manufacturer's instructions.

Northern Blot Analysis

Total RNA was isolated from cell monolayers using an RNA isolation kit from Qiagen Inc. Ten micrograms of total cellular RNA per lane was electrophoresed in a formaldehyde-containing 1.2% agarose gel and transferred to nylon-supported membranes. After baking, membranes were prehybridized overnight in a solution containing 50% formamide, 5× sodium chloride/sodium phosphate/EDTA buffer, 5× Denhardt solution, 0.1% SDS, and 100 μg/mL single-stranded salmon sperm DNA and then hybridized for 12 hours at 42°C with radiolabeled cDNA probes. Probes were labeled with [32P]CTP by random priming. After hybridization, membranes were washed twice for 20 minutes at room temperature in 2× sodium chloride/sodium phosphate/EDTA buffer, 0.1% SDS, twice for 20 minutes in the same solution at 55°C, and twice for 20 minutes in 0.1× sodium chloride/sodium phosphate/EDTA buffer, 0.1% SDS at 55°C. Washed membranes were then subjected to autoradiography.

Transient Transfections

Cells were seeded at a density of 5 × 104 cells/well in 6-well dishes and grown to 50% to 60% confluence. For each well, 2 μg of plasmid DNA was introduced into cells using electroporation by Amaxa protocol (Lonza AG, Basel, Switzerland). After 24 hours of incubation, the medium was replaced with basal medium. The activities of luciferase and β-galactosidase were measured in cellular extract. siRNA transfection was similarly performed.

ChIP Assay

ChIP assay was performed with a kit according to the manufacturer's instructions (Upstate Biotechnology). A total of 1 × 106 cells were cross-linked in a 1% formaldehyde solution for 10 minutes at 37°C. Cells were then lysed in 200 μL of SDS buffer and sonicated to generate 200- to 1000-Bp DNA fragments. After centrifugation, the cleared supernatant was diluted 10-fold with ChIP buffer and incubated with 1.5 μg of the indicated antibody at 4°C. Immune complexes were precipitated, washed, and eluted as recommended. DNA-protein cross-links were reversed by heating at 65°C for 4 hours, and the DNA fragments were purified and dissolved in 50 μL of water. Ten microliters of each sample was used as a template for PCR amplification. ID1 oligonucleotide sequences for PCR primers were: 5′-AGCGGAGAATGCTCCAGCCCAGTTTT-3′ (forward); 5′-AGGCCTCCGAGCAAGCTCTCCCT-3′ (reverse). This primer set encompasses the ID1 promoter segment from nucleotide -932 to -1156 Bp, which includes the EGR-1 and CRE binding sites. PCR was performed at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds for 30 cycles. The PCR products generated from the ChIP template were sequenced, and the identity of the ID1 promoter was confirmed.

Statistical Analysis

The nonparametric Wilcoxon rank-sum test was used for comparing the differences in colon length and histological examination scores between the Id1ΔEndo and Id1fl/fl mice. The generalized linear mixed-effects model was used for evaluating the differences in the probability of having DSS-induced diarrhea or colonic bleeding between the two types of mice. The log-rank test was used for comparing mouse survival between the two types of mice. For other end points, the Student's t-test was used for comparisons between experimental groups. All statistical tests were two-sided. P < 0.05 was considered to be statistically significant.

Results

Id1 Levels Are Increased in Endothelial Cells in Response to Injury

We examined Id1 expression in the mouse colon with a rabbit monoclonal anti-Id1 antibody. Consistent with our previous findings,15 very low levels of Id1 immunoreactivity were detected in the vasculature of normal murine colon; more prominent staining was observed in epithelial stem and transit amplifying cells (Figure 1, A and B). Mouse models of colitis are commonly used for investigating the pathogenesis of IBD. Previously, we reported increased Id1 expression in the colonic epithelium in DSS-induced colitis.15 Here we show that in addition to the up-regulation of Id1 in the epithelium in colitis, Id1 was also increased in the vasculature in the mucosa and submucosa of the inflamed colon (Figure 1, C and D). In an attempt to determine whether there is a similar phenotype in humans, we evaluated ID1 levels in samples from patients with IBD. All patients with IBD had chronic colitis in their surgical-resection specimens. Five (83%) ulcerative colitis patients had moderately to severely active colitis with neutrophilic cryptitis and erosions, and one had mildly active chronic colitis in the examined tissue sections. All of the patients with Crohn disease had chronic active colitis, often with extensive ulceration. Cases of IBD showed strong, diffuse ID1 immunostaining of endothelial cells in areas of inflammation, whereas normal tissues showed an absence of staining in all but one sample (Figure 1, E–J). Two cases each of ulcerative colitis and Crohn disease showed slightly increased staining in inflamed crypts compared with that in normal crypts. To determine whether the observed increase in ID1 expression in endothelial cells would be seen in another inflammatory state, we also assessed ID1 in five cases of acute diverticulitis. In comparison to that in normal mucosa, a significant increase in ID1 immunoreactivity was observed in the endothelium of all five cases of acute diverticulitis. Representative cases are shown in Figure 1, K and L.

Figure 1.

Figure 1

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Localization of inhibitor of DNA binding (ID)-1 on immunohistochemical analysis. A: Hematoxylin and eosin staining in normal mouse colon. B: Expression of Id1 protein in control mouse colon was studied using a rabbit monoclonal Id1 antibody. Id1 staining was undetectable in the majority of vascular endothelial cells (inset, arrowheads). Weak to moderate staining was also noted in epithelial stem and transit amplifying cells (arrow). Inset shows digitally magnified portion of the boxed region. C and D: Mice were given 2.5% dextran sulfate sodium (w/v) for 7 days, followed by 14 days of plain drinking water. Endothelial cells in areas of inflammation show increased Id1 immunostaining (D inset, arrowheads) compared to the normal colon. Injured crypts in areas of colitis also show strong staining for Id1 (arrow). Inset shows digitally magnified portion of the boxed region. E: Normal human colonic mucosa consists of straight, tubular crypts with scattered inflammatory cells in the lamina propria. F: Weak to moderate ID1 staining of numerous crypt epithelial cells is present, although endothelial cells in the lamina propria are negative (arrow). G: Neutrophilic cryptitis in a patient with severe active ulcerative colitis. H: Crypt epithelial cell ID1 staining is slightly increased compared to that in the normal colon. Endothelial staining (arrow) is much stronger in ulcerative colitis than in normal controls. I: An ulcer in Crohn disease contains abundant inflamed granulation tissue with numerous capillaries. J: These dilated, thin-walled vessels are lined by endothelial cells that show strong ID1 immunopositivity (arrow). K: Diverticulitis with inflammatory cells and numerous capillaries. L: Strong ID1 immunoreactivity in endothelial cells lining capillaries in diverticulitis (arrow). Original magnification: ×100 (K and L); ×200 (A–D and J); ×400 (E–H and insets).

PGE2 and TNF-α Induce ID1 Transcription in Endothelial Cells

We next explored potential mechanisms by which ID1 is up-regulated in the endothelium in colitis. Both PGE2 and TNF-α levels are increased in inflamed mucosa and induce ID1 in other cell types.22, 31 Hence, we explored their effects on ID1 expression in cultured endothelial cells. Treatment with PGE2 caused a dose-dependent induction of ID1 mRNA in both human umbilical vein endothelial cells and human intestinal microvascular endothelial cells (Figure 2, A and B). Treatment with PGE2 also induced ID1 protein levels (Figure 2C). To identify the region of the ID1 promoter mediating the inductive effects of PGE2, transient transfections were performed with a series of human ID1 promoter–deletion constructs (Figure 3, A and B). Treatment of human umbilical vein endothelial cells with PGE2 led to more than a fivefold increase in ID1 promoter activity when the -1575 Bp deletion construct was used (Figure 3B). The magnitude of PGE2-mediated induction of ID1 promoter activity remained essentially constant until the -927 Bp deletion construct (5′del 3), which was not stimulated by PGE2. This result implies that one or more promoter elements located between -927 and -1147 Bp are necessary for PGE2-mediated induction of ID1 promoter activity. CREB, E box, and EGR-1 sites are found within this region of the ID1 promoter (Figure 3C). To determine which promoter element(s) were important for mediating the inductive effects of PGE2, transient transfections were performed utilizing ID1 promoter constructs in which these enhancer elements were mutagenized. The induction of ID1 promoter activity by PGE2 was abrogated by mutagenization of the CREB site (Figure 3D). By contrast, mutagenizing the E box or EGR-1 sites had no effect on PGE2-mediated stimulation of ID1 promoter activity. Protein–DNA complexes were immunoprecipitated with an antibody to phospho-CREB, and bound DNA fragments were recovered and subjected to semiquantitative PCR with oligonucleotides specific for the ID1 promoter. The binding of phospho-CREB to the ID1 promoter was enhanced by the treatment of cells with PGE2 (Figure 3E). Finally, silencing of CREB but not EGR-1 blocked PGE2-mediated activation of the ID1 promoter (Figure 3F).

Figure 2.

Figure 2

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Prostaglandin (PG) E2 induces inhibitor of DNA binding (ID)-1 in human endothelial cells. Endothelial cells were treated with vehicle or the indicated concentration of PGE2 for 24 hours. A–C: Human umbilical vein endothelial cells (HUVECs, A and C) and human intestinal microvascular endothelial cells (HIMECs, B). A and B: Total cellular RNA was isolated from cells. Ten micrograms of RNA was added to each lane and subjected to Northern blot analysis. The blots were hybridized with probes that recognized ID1 and β-actin. C: Cellular protein (100 μg/lane) was subjected to immunoblot analysis. The blot was probed with antibodies to ID1 and β-actin.

Figure 3.

Figure 3

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cAMP-response element–binding (CREB) protein site is important for prostaglandin (PG) E2–mediated stimulation of ID1 promoter activity. A: The different human ID1 promoter deletion constructs used for transfection analyses. B: Human umbilical vein endothelial cells (HUVECs) were transfected with 1.8 μg of a series of human ID1 promoter–luciferase deletion constructs [1.5 basic vector (BV); 5′del 1 to 7] and 0.2 μg of pSVβgal. C: Nucleotide sequence of human ID1 promoter. D: Cells were transfected with 1.8 μg of ID1 promoter–luciferase (-1147/-937) or -1147/-937 Bp ID1 promoter construct in which the E box (mEbox), CREB (mCREB), or early growth response 1 (mEGR-1) sites were mutagenized. Cells also received 0.2 μg of pSVβgal. E: Chromatin immunoprecipitation assays. HUVEC cells were treated with vehicle or 0.5 μmol/L PGE2 for 3 hours. Chromatin fragments were immunoprecipitated with pCREB antibody, and the ID1 promoter region was amplified by PCR. DNA was sequenced, and the PCR product was confirmed to be the ID1 promoter. The ID1 promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step. F: HUVEC cells were transfected with 0.9 μg of ID1 promoter construct and 0.2 μg of pSVβgal. The column labeled Control siRNA represents cells that also received 0.9 μg of siRNA to Green fluorescent protein; the column labeled CREB siRNA represents cells that received 0.9 μg of siRNA to CREB; the column labeled EGR-1 siRNA represents cells that received 0.9 μg of siRNA to EGR-1. The total amount of transfected DNA for each condition was kept constant at 2 μg by using corresponding empty expression vector. In B, D, and F, 24 hours after transfection, the cells were treated with vehicle (control) or 0.5 μmol/L PGE2. Twenty-four hours after treatment, cellular lysates (100 μg/lane) were isolated and subjected to either Western blot analysis (F, top panel) or measurements of reporter activities (B, D, and bottom panel of F). In F, the blots were probed as indicated with antibodies to CREB, EGR-1, and β-actin. In B, D, and F, luciferase activity represents data that have been normalized to β-galactosidase activity. Data are expressed as means ± SD. n = 6. ∗∗∗P < 0.001 versus cells transfected with control siRNA. AP, activator protein; ATF, activating transcription factor; YY, yin and yang.

In addition to PGE2, levels of TNF-α are also increased in IBD.6, 7 Hence, it was of interest to determine whether this proinflammatory cytokine could induce ID1 in endothelial cells. TNF-α caused dose-dependent induction of ID1 mRNA in both human umbilical vein endothelial cells and human intestinal microvascular endothelial cells (Figure 4, A and B). ID1 protein was also induced by TNF-α (Figure 4C). We next identified the region of the ID1 promoter that was important for mediating the inductive effects of TNF-α. Treatment of human umbilical vein endothelial cells with TNF-α led to a several-fold increase in ID1 promoter activity when the full-length construct (1.5 basic vector) was used (Figure 5A). The magnitude of TNF-α–mediated induction of ID1 promoter activity was constant until the -927 Bp deletion construct (5′del 3) was used. To determine which promoter element(s) were important for mediating the inductive effects of TNF-α, transient transfections were performed utilizing ID1 promoter constructs in which the enhancer elements between -927 and -1147 (see previous paragraph) were mutagenized. The induction of ID1 promoter activity by TNF-α was abrogated by mutagenizing the EGR-1 site (Figure 5B). By contrast, mutagenization of the E box or CREB site had no effect on TNF-α–mediated stimulation of ID1 promoter activity (Figure 5B). To further evaluate the role of EGR-1, ChIP assays were performed. The binding of EGR-1 to the ID1 promoter was enhanced by the treatment of cells with TNF-α (Figure 5C). Importantly, silencing of EGR-1 but not CREB blocked TNF-α–mediated stimulation of ID1 promoter activity (Figure 5D).

Figure 4.

Figure 4

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Tumor necrosis factor (TNF)-α induces inhibitor of DNA binding (ID)-1 in human endothelial cells. Human umbilical vein endothelial cells (HUVECs, A and C) and human intestinal microvascular endothelial cells (HIMECs, B) were treated with vehicle or with the indicated concentration of TNF-α for 24 hours. A and B: Total cellular RNA was isolated from cells. Ten micrograms of RNA was added to each lane and subjected to Northern blot analysis. The blots were hybridized with probes that recognized ID1 and β-actin. C: Cellular protein (100 μg/lane) was subjected to immunoblot analysis. The blot was probed with antibodies to ID1 and β-actin, respectively.

Figure 5.

Figure 5

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Early growth response (EGR)-1 binding site is important for tumor necrosis factor (TNF)-α–mediated stimulation of ID1 promoter activity. A: Human umbilical vein endothelial cells (HUVECs) were transfected with 1.8 μg of a series of human ID1 promoter–luciferase deletion constructs [1.5 basic vector (BV), 5′del 1 to 7] and 0.2 μg of pSVβgal. B: Cells were transfected with 1.8 μg of ID1 promoter–luciferase (-1147/-937) or the -1147/-937 Bp ID1 promoter construct in which the cAMP response element–binding protein (mCREB), E box (mEbox), or EGR-1 (mEGR-1) sites were mutagenized. Cells also received 0.2 μg of pSVβgal. C: Chromatin immunoprecipitation assays. HUVECs were treated with vehicle or 10 ng/mL TNF-α for 3 hours. Chromatin fragments were immunoprecipitated with an antibody to EGR-1, and the ID1 promoter region was amplified by PCR. DNA was sequenced, and the PCR product was confirmed to be the ID1 promoter. The ID1 promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step. D: HUVECs were transfected with 0.9 μg of ID1 promoter construct and 0.2 μg of pSVβgal. The column labeled Control siRNA represents cells that also received 0.9 μg of siRNA to green fluorescent protein; the column labeled EGR-1 siRNA represents cells that received 0.9 μg of siRNA to EGR-1; the column labeled CREB siRNA represents cells that received 0.9 μg of siRNA to CREB. The total amount of DNA transfected for each condition was kept constant at 2 μg by using corresponding empty expression vector. In A, B, and D, 24 hours after transfection, the cells were treated with vehicle (control) or 10 ng/mL TNF-α. Twenty-four hours after treatment, cellular lysates were isolated and reporter activities measured. Luciferase activity represents data that have been normalized to β-galactosidase activity. Data are expressed as means ± SD. n = 6. ∗∗∗P < 0.001 versus cells transfected with GFP siRNA.

Endothelial Specific Depletion of ID1 Increases the Severity of Colitis

In previous work,15 we found that both the severity of colitis and mortality were greater when ID1 was completely knocked out as opposed to being selectively deleted in epithelial cells. This finding suggests that in addition to the important role of ID1 in the stem/progenitor compartment, endothelial ID1 may be involved in the pathogenesis of colitis. To determine whether vascular ID1 is also functionally important for intestinal homeostasis in response to injury, we generated endothelium-specific Id1 knockout (Id1ΔEndo) mice by crossing Id1fl/fl mice with Cdh5-CreERT2 transgenic mice, which expressed a tamoxifen-inducible form of CreERT2 under the control of endothelial-specific VE-cadherin regulatory sequences.23 Treatment with DSS ad libitum over a 7-day period, followed by regular drinking water for 14 days, led to a marked increase in Id1 in the colonic epithelium and endothelium of Id1fl/fl control mice (Figure 6A). By contrast, Id1 was induced in the colonic epithelium only in the Id1ΔEndo mice after similar treatment (Figure 6A). To evaluate whether the loss of Id1 in the vasculature affected the morphology of the blood vessels, CD31 (platelet endothelial cell adhesion molecule) and α smooth muscle actin IHC analysis was performed (Figure 6, B and C). Mice treated with DSS exhibited frequent CD31+ aggregates, a phenotype previously found in the tumor vasculature of Id1 knockout mice.32 These aggregates also stained strongly for α smooth muscle actin (Figure 6C) but not LYVE 1 (data not shown), a marker of lymphatic endothelium. No such aggregates were observed in nontreated mice, suggesting that this phenotype is observed only in the context of injury. Notably, these aggregates were more abundant in DSS-treated Id1ΔEndo mice than in control mice (Figure 6D).

Figure 6.

Figure 6

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Id1 deletion results in morphological and vascular functional changes in the colon. Mice were given plain drinking water or 2.5% dextran sulfate sodium (DSS) (w/v) for 7 days, followed by 14 days of plain drinking water. A: The increased expression of inhibitor of DNA binding (Id)-1 in endothelial cells (arrow) in colitis (Id1fl/fl) is abrogated in endothelium-specific Id1 knockout (Id1ΔEndo) mice. By contrast, colitis is associated with increased Id1 in the epithelium in both Id1fl/fl and Id1ΔEndo mice. B: CD31 immunohistochemical (IHC) analysis of colonic sections from Id1ΔEndo mice in the presence of DSS-induced colitis. Arrow indicates a CD31+ aggregate. Inset shows digitally magnified portion of the boxed region. C: α Smooth muscle actin (Sma) IHC analysis on adjacent section. Arrow indicates location of the CD31+ aggregate shown in B. Inset shows digitally magnified portion of the boxed region. D: Quantification of CD31+ aggregates in Id1fl/fl and Id1ΔEndo mice under basal conditions and in the presence of DSS-induced colitis. E: Vascular permeability was determined by measuring Evans blue (EB) dye extravasation in wild-type (WT) and Id1−/− (KO) mice under basal conditions and in the presence of DSS colitis. F: Hypoxia inducible factor (Hif)-1α IHC analysis; staining for Hif-1α was performed on a section adjacent to that shown in B and C. Arrow indicates location of the CD31+ aggregate shown in B. Inset shows digitally magnified portion of the boxed area. Crypts proximal to the CD31+ aggregate stained positive for Hif-1α (arrowheads), whereas tissues distal to the CD31+ aggregate were Hif-1α–negative (asterisks). Data are expressed as means ± SD. n = 3 per group (D, control); n = 4 to 6 per group (E); n = 14 per group (D, DSS). ∗∗P < 0.01, ∗∗∗P < 0.001. Original magnification: ×200 (A–C and F); ×400 (insets).

To determine whether Id1 loss was associated with functional changes in the vasculature, colonic vascular permeability was determined by quantifying Evans blue dye extravasation. Id1 deficiency led to a small increase in Evans blue dye extravasation (Figure 6E). DSS-induced colitis led to an increase in extravasation, with a greater effect observed in Id1-deficient mice than in wild-type mice. Theoretically, ID1-dependent changes in vascular permeability might result in local hypoxia. We tested this possibility by performing IHC analysis for Hif-1α. Interestingly, Hif-1α levels were increased in colon crypts proximal, to but not distal to, the CD31+ aggregates (Figure 6F). We also assessed the severity of colitis. Id1ΔEndo mice exhibited more severe colitis, including more severe diarrhea and bleeding, shorter colons, and worse findings on histological examination compared with those in Id1fl/fl mice (Figure 7, A–D). Consistent with having more severe colitis, Id1ΔEndo mice also had a strong trend toward reduced survival compared with that in the control mice (P = 0.06) (Figure 7E). Taken together, these results imply that endothelial ID1 is required for a normal response to colonic injury.

Figure 7.

Figure 7

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Id1 deletion in endothelial cells sensitizes mice to more severe colitis. Mice were given plain drinking water or 2.5% dextran sulfate sodium (w/v) for 7 days, followed by 14 days of plain drinking water. A–E: Percentages of mice with diarrhea (A), bleeding (B), colon length (C), histological examination score (D), and mortality over time (E). ∗P < 0.05, ∗∗P < 0.01. n = 14 per group (D); n = 28 to 31 per group (A and B); n = 46 to 49 per group (C); n = 55 to 59 per group (E).

Discussion

In inflamed mucosa in patients with IBD, active angiogenesis occurs.4, 33, 34 Several proangiogenic factors, including vascular endothelial growth factor (VEGF), IL-8, PGE2, and fibroblast growth factor have been shown to be overexpressed in Crohn disease and ulcerative colitis.8, 35, 36, 37 However, the significance of ID1, a proangiogenic inhibitory transcription factor in regulating endothelial function, in IBD has not been studied. In the present report, we show that Id1 levels are increased in the endothelium in both experimental colitis and human IBD. The induction of ID1 in the endothelium appears to be important for protecting the colonic epithelium from injury as endothelial cell-specific deletion of Id1 leads to more severe colitis by multiple clinical and pathological measures (Figure 7). Based on our finding that ID1 is also up-regulated in the endothelium in diverticulitis, another inflammatory state, it is possible that this is an adaptive homeostatic mechanism that is relevant in multiple forms of mucosal injury.

Levels of PGE2 and TNF-α are increased in inflamed mucosa in IBD patients.6, 7, 8 Both of these molecules have been reported to induce ID1 in other cell types.22, 31 Hence, we investigated whether either PGE2 or TNF-α could induce ID1 in endothelial cells. Treatment with PGE2 induced ID1 transcription in human endothelial cells. Several findings support a role for CREB in mediating the induction of ID1 by PGE2. Increased binding of pCREB to the ID1 promoter was detected in PGE2-treated cells. Activation of the ID1 promoter by PGE2 was abrogated when the CRE was mutagenized. Finally, PGE2-mediated induction of ID1 promoter activity was attenuated by the silencing of CREB. These findings are consistent with those from prior studies that have shown a role for CREB in mediating the activation of gene expression by PGE2.17, 38 Interestingly, TNF-α induced ID1 by a different mechanism. TNF-α stimulated the binding of EGR-1 to the ID1 promoter. Moreover, TNF-α–mediated activation of the ID1 promoter was blocked by mutagenization of the EGR-1 site in the ID1 promoter or silencing EGR-1. This result fits with prior observations that TNF-α can stimulate gene expression via EGR-1.39 Moreover, levels of EGR-1 are increased in the inflamed mucosa of IBD patients.39 Given the observed differences in signaling mechanisms that lead to ID1 induction, it would be of interest to determine whether the combination of PGE2 and TNF-α has additive or synergistic effects.

Our results may also provide insight into the mechanism by which nonsteroidal anti-inflammatory drugs, prototypic inhibitors of PGE synthesis, can exacerbate IBD.40, 41 Based on our finding that PGE2 induces ID1 in endothelial cells, it is possible that nonsteroidal anti-inflammatory drugs prevent the up-regulation of vascular ID1 in colitis, thereby leading to more severe disease. Loss of Id1 is shown here to enhance the frequency of CD31+ aggregates, increase vessel leakiness, and enhance Hif-1α expression adjacent to the CD31+ aggregates in experimental colitis. The increase in vessel leakiness might be the result of Hif-1α–mediated induction of the vascular permeability factor Vegf.42 The current findings are consistent with those from prior studies that have demonstrated that a loss of Id1 can lead to structural and functional vascular abnormalities in tumor-bearing mice.19, 32, 43 However, the present study could not distinguish whether the observed vascular abnormalities are a consequence or a cause of the more severe colitis. Nonetheless, our data suggest that the depletion of ID1 in the endothelium contributes to hypoxia, which can exacerbate inflammation.44

In addition to increased PGE2 signaling in the inflamed mucosa, other factors such as TNF-α might contribute to the increase in ID1 expression in the endothelium of the microvasculature. VEGF itself has been shown to lead to a dramatic up-regulation of ID1 in the bone marrow.45 Importantly, the consequences of Id1 loss on microvessel integrity in the cancer setting can be rescued with wild-type bone marrow transplantation in otherwise Id1-deficient mice. It will be of interest, therefore, to determine in the IBD setting whether ID1-expressing cells derived from the bone marrow reach the inflamed colon and whether these cells are of functional consequence in response to tissue injury. Although anti-VEGF therapy has been reported to ameliorate experimental colitis in association with reducing excessive vascular permeability,46 it may also have the unintended consequence of reducing the mobilization of ID1+ cells from the bone marrow and thereby limiting the effectiveness of the therapy. Combining anti-VEGF therapy with ID1+ bone marrow–derived cells may therefore yield significant clinical benefits.

Acknowledgments

We thank the Molecular Cytology Core Facility at Memorial Sloan Kettering for assistance with IHC analysis and Dr. Tim Hla (New York, NY) for assistance with data evaluation.

Footnotes

Supported by a grant from the New York Crohn's Foundation (A.J.D.) and NIH grant UL1TR000457 from the Clinical and Translational Science Center, Weill Cornell Medical College (X.K.Z.). The Molecular Cytology Core Facility, Memorial Sloan Kettering, was supported by NIH/NCI grant P30 CA008748.

Disclosures: R.B. is the chief science officer, chair of the Scientific Advisory Board, and owns shares in Angiogenex, a company devoted to antagonizing ID protein activity in human patients.

Contributor Information

Robert Benezra, Email: benezrar@mskcc.org.

Andrew J. Dannenberg, Email: ajdannen@med.cornell.edu.

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