Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli

Abstract

Background & aims: Gastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP) signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5^+ve cells during inflammation.

Methods: The H⁺/K⁺-adenosine triphosphatase β-subunit promoter was used to express the BMP inhibitor noggin (Nog) in the stomach (H⁺/K⁺-Nog mice). Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre) mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1a^flox/flox mice). Lgr5/GFP^+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom). Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H⁺/K⁺-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction.

Results: Lgr5-Cre;Bmpr1a^flox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP^+ve cells. Infection of Lgr5-Cre;Bmpr1a^flox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato^+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, and with anti-IF, -CD44, -CD44v9, and -bromodeoxyuridine antibodies.

Conclusions: Inflammation and inhibition of BMP signaling activate Lgr5^+ve cells, which give rise to metaplastic, dysplastic, proliferating lineages that express markers of mucus neck and zymogenic cell differentiation.

Keywords: ATPase, adenosine triphosphatase; BMP, bone morphogenetic protein; BrdU, bromodeoxyuridine; Chief Cells; Differentiation; Dysplasia; EGFP, enhanced green fluorescent protein; ERK, extracellular signal–regulated kinase; GFP, green fluorescent protein; GSII, Griffonia (Bandeiraea) simplicifolia lectin II; H/K-nog, H/K-noggin; HBSS, Hank's balanced salt solution; IF, intrinsic factor; Metaplasia; QRT-PCR, quantitative reverse-transcription polymerase chain reaction; SPEM, spasmolytic polypeptide expressing metaplasia; TFF2, Trefoil factor 2; mRNA, messenger RNA.

Figure 1

Deletion of Bmpr1a in Lgr5 cells. GFP^+ve cells from 1- to 2-month-old Lgr5-EGFP-ires-CreERT2 mice (Lgr5-Cre mice) and Lgr5-EGFP-ires-CreERT2;Bmpr1a^flox-flox mice (Lgr5-Cre;Bmpr1a^flox-flox mice) were isolated by flow cytometry. Mice were treated with 1 intraperitoneal. injection of tamoxifen (0.1 mg/g body weight) and killed 15 days after tamoxifen. Flow cytometry plots of cells isolated from (A) Lgr5-Cre mice and (B) Lgr5-Cre;Bmpr1a^flox-flox mice. (C) Flow cytometry plot of cells isolated from nontransgenic negative control mice. (D) Bmpr1a mRNA signals in cells from Lgr5-Cre;Bmpr1a^flox-flox mice were compared with those detected in cells from control mice using QRT-PCR. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. *P < .05.

Figure 2

Expression of Bmpr1a, Id1, and Lgr5 in the lesser and greater curvatures of Lgr5-Cre and Lgr5-Cre;Bmpr1a^flox-floxmice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and killed 5 months after tamoxifen. Bmpr1a, Id1, and Lgr5 mRNA signals in the lesser and greater curvatures of Lgr5-Cre mice were compared with those detected in Lgr5-Cre;Bmpr1a^flox-flox mice using QRT-PCR. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. *P < .05.

Figure 3

Regulation of cells with Lgr5 transcriptional activity by BMP signaling. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis for 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. (A) Paraffin sections of the lesser and greater curvatures of Lgr5-Cre and Lgr5-Cre;Bmpr1a^flox-flox mice were stained with Alexa 488–conjugated anti-GFP antibodies (green) together with an anti-H⁺,K⁺-ATPase α-subunit primary antibody and an Alexa 555–conjugated secondary antibody (red). Scalebar: 50 μm. (C) Paraffin sections of the lesser curvature of Lgr5-Cre and Lgr5-Cre;Bmpr1a^flox-flox mice were stained with Alexa 488–conjugated anti-GFP antibodies (green) together with an anti-Ki67 antibody and an Alexa 555–conjugated secondary antibody (red). Scale bar: 20 μm. Arrows point to GFP/Ki67-positive cells. Bars represent the number of (B) GFP- and of (D) GFP/Ki67-positive cells that were detected in the mucosa of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis (H. f.). Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. *P < .05 vs Lgr5-Cre mice. **P < .05 vs Lgr5-Cre;Bmpr1a^flox-flox mice. DAPI, 4'6-diamidino-2-phenylindole.

Figure 4

Expression of IF in cells with Lgr5 transcriptional activity. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis (H. f.) were stained with anti-IF primary antibodies and an Alexa 555–conjugated secondary antibody (red) together with Alexa 488–conjugated anti-GFP antibodies (green). Scale bar: 25 μm. Similar results were observed in at least 1 other mouse and in 6 other mice in the H felis–infected Lgr5-Cre;Bmpr1a^flox-flox group. DAPI, 4'6-diamidino-2-phenylindole.

Figure 5

Enhanced inflammatory changes, metaplasia, and dysplasia in the gastric epithelium of H felis–infected Lgr5-Cre;Bmpr1a^flox-floxmice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. (A) Representative H&E-stained paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis (H. f.). The magnified window depicts dysplastic and inflammatory changes in H felis–infected Lgr5-Cre;Bmpr1a^flox-flox mice. Scale bars: (A) 100 μm, (B) 25 μm. (B) Red arrows point to areas of dysplastic epithelium, black arrows indicate clusters of polymorphonuclear cells. (C) Graph bars represent the percentage of fields affected by gastritis, neutrophilic infiltrates, metaplasia, and dysplasia calculated in both Lgr5-Cre and Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. *P < .05 vs H felis–infected Lgr5-Cre mice.

Figure 6

Decreased parietal cells and increased cell proliferation in Helicobacter-infected Lgr5-Cre;Bmpr1a^flox-floxmice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a^flox-flox mice, in the presence and absence of H felis (H. f.), were stained with an (A) anti-H⁺,K⁺-ATPase α-subunit primary antibody and an Alexa 488–conjugated secondary antibody (green), and with an (C) anti-Ki67 primary antibody and an Alexa 555–conjugated secondary antibody (red). Scale bars: 100 μm. Bars represent the number of (B) H⁺,K⁺-ATPase α-subunit and of (D) Ki67-positive cells detected in Lgr5-Cre mice and in Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis. Values are shown as means ± SE. Numbers in parenthesis indicate the number of animals used in each group. *P < .05 vs Lgr5-Cre mice. **P < .05 vs Lgr5-Cre;Bmpr1a^flox-flox mice. DAPI, 4'6-diamidino-2-phenylindole.

Figure 7

Development of SPEM in H felis–infected Lgr5-Cre;Bmpr1a^flox-floxmice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of Lgr5-Cre and of Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis (H. f.) were stained with anti-IF primary antibodies and Alexa 555–conjugated secondary antibodies (red) together with Alexa 488–conjugated GS II (green). Gastric paraffin sections of the lesser curvature of H felis–infected Lgr5-Cre;Bmpr1a^flox-flox mice were stained with anti-TFF2 primary antibodies and an Alexa 488–conjugated secondary antibody. Scale bar: 100 μm. Similar results were observed in at least 3 other mice in each group. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 8

Expression of CD44 in H felis–infected Lgr5-Cre;Bmpr1a^flox-floxmice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. (A) Paraffin sections of the lesser curvature of Lgr5-Cre and Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis (H. f.) stained with anti-CD44 primary antibodies and a biotin-conjugated secondary antibody. The magnified window shows intense CD44 staining of epithelial cells in H felis–infected Lgr5-Cre;Bmpr1a^flox-flox mice. Scale bar: 100 μm, 50 μm in the magnified window. Arrows point to CD44^+ve cells. (B) Paraffin sections of the lesser curvature of noninfected and H felis–infected Lgr5-Cre;Bmpr1a^flox-flox mice stained with anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies. The magnified window shows CD44v9 staining of epithelial cells at the base of glands in H felis–infected Lgr5-Cre;Bmpr1a^flox-flox mice. Scale bar: 100 μm, 50 μm in the magnified window. Arrows point at CD44v9^+ve cells. (C) Paraffin sections of the lesser curvature of H felis–infected Lgr5-Cre;Bmpr1a^flox-flox mice stained with anti-CD44 primary antibodies and Alexa 594-conjugated secondary antibodies (red) together with Alexa 488–conjugated GSII (green), anti-CD44 primary antibodies and Alexa 488–conjugated secondary antibodies (green) together with IF antibodies and Alexa 555–conjugated secondary antibodies (red) and anti-CD44 primary antibodies and Alexa 488–conjugated secondary antibodies (green) together with anti-Ki67 antibodies and Alexa 555–conjugated secondary antibodies (red). Similar results were observed in at least 2 other mice in each group. Scale bar: 50 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 9

Expression of Cd44 and c-Myc mRNAs in the lesser curvature of Lgr5-Cre;Bmpr1a^flox-floxmice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a^flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Cd44 and c-Myc mRNA signals in samples of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a^flox-flox mice in the presence and absence of H felis (H. f.) were measured using QRT-PCR. Numbers in parenthesis indicate the number of animals used in each group. *P < .05.

Figure 10

Distribution of tomato-positive cells in H felis–infected Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-Tom mice. One- to 2-month-old Lgr5-Cre;Rosa26-Tom mice and Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of Lgr5-Cre;Rosa26-Tom and Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom in the presence and absence of H felis (H. f.) were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies. Matching sections of the gastric mucosa of both noninfected and H felis–infected Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom mice were stained with H&E. Similar results were observed in at least 3 other mice in each group. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 11

Lineage tracing of cells with Lgr5 transcriptional activity in H felis–infected Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-Tom mice. 1-2-month old Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom mice were treated with one i.p. injection of tamoxifen (0.1 mg/g body weight) and inoculated with H. felis two months post tamoxifen. Animals were analyzed 3 months after inoculation, and 5 months after tamoxifen injection. Gastric paraffin sections of the lesser curvature of H felis–infected Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom mice were stained with anti-tomato (red fluorescent protein) primary antibodies and Alexa 555–conjugated secondary antibodies together with anti-IF antibodies and Alexa 488–conjugated secondary antibodies (IF/Tom), Alexa 488–conjugated GSII (GSII/Tom), Alexa 488–conjugated Ulex europaeus agglutinin 1 (UEA1), anti-CD44 and anti-CD44v9 primary antibodies and Alexa 488–conjugated secondary antibodies (CD44/Tom and CD44v9/Tom), and anti-BrdU primary antibodies and Alexa 488–conjugated secondary antibodies (BrdU/Tom). Scale bars: 50 μm. Similar results were observed in at least 5 other H felis–infected Lgr5-Cre;H⁺/K⁺-Nog;Rosa26-tdTom mice except for the experiments with the anti-CD44v9 antibodies that were repeated in 1 other mouse.