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. 2023 Dec 20:14:1323865.
doi: 10.3389/fphys.2023.1323865. eCollection 2023.

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

Mariana Sponchiado  1 , Angelina L Bonilla  1 , Luz Mata  1 , Kalene Jasso-Johnson  1 , Yan-Shin J Liao  1 , Amy Fagan  1 , Victor Moncada  1 , Leah R Reznikov  1
Affiliations

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

Mariana Sponchiado et al. Front Physiol. .

Abstract

Introduction: Club cells are precursors for mucus-producing goblet cells. Interleukin 1β (IL-1B) is an inflammatory mediator with pro-mucin activities that increases the number of mucus-producing goblet cells. IL-1B-mediated mucin production in alveolar adenocarcinoma cells requires activation of the cAMP response element-binding protein (CREB). Whether the pro-mucin activities of IL-1B require club cell CREB is unknown. Methods: We challenged male mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-1B or vehicle. Secondarily, we studied human "club cell-like" H322 cells. Results: IL-1B increased whole lung mRNA of secreted (Mucin 5ac, Mucin 5b) and tethered (Mucin 1, Mucin 4) mucins independent of genotype. However, loss of club cell Creb1 increased whole lung mRNA of member RAS oncogene family (Rab3D), decreased mRNA of the muscarinic receptor 3 (M3R) and prevented IL-1B mediated increases in purinergic receptor P2Y, (P2ry2) mRNA. IL-1B increased the density of goblet cells containing neutral mucins in wildtype mice but not in mice with loss of club cell Creb1. These findings suggested that club cell Creb1 regulated mucin secretion. Loss of club cell Creb1 also prevented IL-1B-mediated impairments in airway mechanics. Four days of pharmacologic CREB inhibition in H322 cells increased mRNA abundance of forkhead box A2 (FOXA2), a repressor of goblet cell expansion, and decreased mRNA expression of SAM pointed domain containing ETS transcription factor (SPDEF), a driver of goblet cell expansion. Chromatin immunoprecipitation demonstrated that CREB directly bound to the promoter region of FOXA2, but not to the promoter region of SPDEF. Treatment of H322 cells with IL-1B increased cAMP levels, providing a direct link between IL-1B and CREB signaling. Conclusion: Our findings suggest that club cell Creb1 regulates the pro-mucin properties of IL-1B through pathways likely involving FOXA2.

Keywords: FOXA2; IL-1B; SPDEF; club cell; cystic fibrosis; mucin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

FIGURE 1
FIGURE 1
Percentage of club cells expressing Creb1 is increased following repeated administration of IL-1B. (A) Representative images of dual label immunohistochemistry for Creb1 (brown) and clara cell secretory protein (red) in airway cross sections from Creb1fl/flScgb1a1cre or Creb1fl/flScgb1a1wt mice. Arrow indicates an example of CCSP-positive (club) cell expressing presumably nuclear staining for Creb1. Scale bar is 20 μm. (B) Percentage of club cells expressing Creb1. * = compared to vehicle control, p = 0.0013; ** = main genotype effect across treatments, p = 0.0055. (C) Density of club cells in airway. For B and C, individual points are data collected from a single mouse. n = 5 per group. Abbreviations: WT, wild type; Scgb1a1cre, club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle.
FIGURE 2
FIGURE 2
IL-1B treatment increases mRNA abundance of secreted and tethered mucins. Abundance of mRNA in the murine lung for (A) Muc5ac, * = main effect of IL-1B treatment across genotype, p = 0.0246; (B) Muc5b, * = main effect of IL-1B treatment across genotype, p = 0.0342; (C) Muc1, * = main effect of IL-1B treatment across genotype, p = 0.0206; and (D) Muc4 * = main effect of IL-1B treatment across genotype, p = 0.0048. Bronchoalveolar lavage concentrations of (E) Muc5ac and (F) Muc5B. A trend for statistical significance for main genotype effect across treatment was observed for Muc5ac concentrations (p = 0.054). For all panels, individual points are data collected from a single mouse: Creb1fl/flScgb1a1wt mice treated with vehicle (n = 6) or IL-1B (n = 7); Creb1fl/flScgb1a1cre mice treated with vehicle (n = 8) or IL-1B (n = 8). For panel E and F, a single mouse from the Creb1fl/flScgb1a1cre treated with vehicle group was a statistical outlier using Grubbs outlier test. Therefore, concentrations of Muc5ac and Muc5b from this mouse were not included. Abbreviations: WT, wild type; Scgb1a1cre, club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle.
FIGURE 3
FIGURE 3
Conditional loss of club cell Creb1 modifies mucin secretion characteristics. (A) Abundance of mRNA in the murine lung for (A) Rab3D, * = main effect of genotype across treatment, p = 0.0280; (B) P2ry2, * = compared to vehicle control, p = 0.0233; Significant genotype x treatment interaction, p = 0.0142; (C) M3R, * = main effect of genotype across treatment, p = 0.0179; and (D) Muc4, * = main effect of IL-1B treatment across genotype, p = 0. 0048. (D) Density of goblet cells staining for Alcian-Blue/PAS under basal and methacholine stimulated conditions. Density is for cells with acidic mucins, as detected by dark blue staining. Main effect of methacholine, p < 0.0001. (E) Density of goblet cells staining for Alcian-Blue/PAS under basal and methacholine stimulated conditions. Density is for cells with neutral mucins, as detected by purple/magenta staining. Main effect of methacholine, p = 0.0036. Significant treatment x genotype interaction, p = 0.0069. * = compared to Creb1fl/flScgb1a1wt vehicle control under methacholine stimulated conditions, p = 0.0028; ** = to Creb1fl/flScgb1a1wt IL-1B under methacholine stimulated conditions, p = 0.0067. Post methacholine bronchoalveolar lavage concentrations of (F) Muc5ac and (G) Muc5b. (H) Mean immunohistochemistry signal intensity of Muc5b in the airway post methacholine stimulation. For all panels, individual points are data collected from a single mouse. For Panel F, one mouse from the Creb1fl/flScgb1a1cre + VEH group had Muc5ac concentrations below detection and was therefore not included in analysis. Abbreviations: WT, wild type; Scgb1a1cre, club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle; MCh, methacholine.
FIGURE 4
FIGURE 4
Conditional loss of club cell Creb1 diminishes IL-1B-mediated alterations in pulmonary mechanics. Basal airway resistance (A), airway elastance (C), tissue damping (E) and tissue elastance (G) as measured with a forced oscillation technique. Methacholine dose response curves normalized to basal values for airway resistance (B), airway elastance (D), tissue damping (F) and tissue elastance (H). For Panels (A, C, E, G), individual points are data collected from a single mouse. For Panel (B), * = main effect of IL-1B treatment in wildtype mice compared to wild type vehicle controls, p = 0.0005. For Panel (D), * = main effect of IL-1B treatment in wildtype mice compared to wild type vehicle controls, p < 0.0001. For Panel (F), * = main effect of IL-1B treatment in wildtype mice compared to wild type vehicle controls, p < 0.0001. For Panel (H), a significant genotype x treatment x methacholine dose interaction was noted, allowing for individual post hoc comparisons to be conducted. * = IL-1B treatment in wildtype mice compared to wild type vehicle controls; at 50 mg/mL, p = 0.0351; at 100 mg/mL, p < 0.0001. Legend key in Panel (B) applies to Panels (D, F, H). n = 7 for Creb1fl/flScgb1a1wt mice treated with vehicle or IL-1B; n = 9 for Creb1fl/flScgb1a1cre mice treated with vehicle or IL-1B. Abbreviations: WT, wild type; Scgb1a1cre, club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle.
FIGURE 5
FIGURE 5
Conditional loss of club cell Creb1 modulates inflammatory responses post methacholine. (A) Numbers of cells in the BAL. (B) Percentage of cells that were granulocytes in the BAL. * = main effect of genotype across treatments, p = 0.0418. (C) Expression of lung Il1r1 mRNA normalized to vehicle control wild type mice. *p = 0.0324. (D). Heat map of genes that were differentially expressed in the murine lung due to treatment and/or genotype. Scale represents fold change. More details are available in Supplementary Table S4. For Panels (A–C), n = 7 for Creb1fl/flScgb1a1wt mice treated with vehicle or IL-1B; n = 9 for Creb1fl/flScgb1a1cre mice treated with vehicle or IL-1B. For Panel (D), n = 6 mice per group. Abbreviations: WT, wild type; Scgb1a1cre, club cell promoter driving CRE recombinase; IL-1B, Interleukin 1β; VEH, vehicle. Csf2, granulocyte macrophage colony stimulating factor; Il10, interleukin 10; Ly96, myeloid differentiation factor 2; Mpo, myeloperoxidase; Stat4, signal transducer and activator of transcription 4; Tlr8, toll like receptor 8.
FIGURE 6
FIGURE 6
CREB regulates transcripts necessary for goblet cell expansion in human cell line reportedly consisting of club cells. (A) Abundance of CREB1 mRNA in response to IL-1B and/or CREB inhibitor. * = compared to respective vehicle-treated cells, p < 0.05. (B) Abundance of BDNF mRNA; * = main effect of CREB inhibition across treatment, p = 0.0033. (C) Abundance of FOXA2 mRNA; * = main effect of CREB inhibition across treatment, p < 0.0001. (D) Abundance of SPDEF mRNA; * = main effect of CREB inhibition across treatment, p < 0.0001. (E) Abundance of FOXA2 mRNA in cells treated with recombinant CREB protein; * = compared to vehicle control, p = 0.0480. (F) Abundance of SPDEF mRNA in cells treated with recombinant CREB protein; * = compared to vehicle control, p = 0.0371. Abbreviations: IL-1B, Interleukin 1β; VEH, vehicle; CREB1, cAMP responsive element binding protein 1; BDNF, brain derived neurotrophic factor; FOXA2, forkhead box A2; SPDEF, SAM pointed domain containing ETS transcription factor.
FIGURE 7
FIGURE 7
IL-1B increases cAMP concentrations and CREB directly interacts with FOXA2. (A) Forskolin-treated NCI-H322 cells were subjected to ChIP assays using primers designed encompassing CRE sites (binding site for CREB). End-point PCR was used to assess template DNA amplification in CREB or IgG immunoprecipitated samples. Genomic DNA was used as positive control for the PCR reactions. (B) Abundance of IL1R1 mRNA in H322 cells treated with IL-1B and/or CREB inhibitor. * = main effect of IL-1B treatment across groups, p = 0.0035. (C) Concentrations of cyclic AMP (cAMP) in H322 cells treated with IL-1B for 8 h. * = compared to vehicle control, p < 0.0001. (D) Proposed mechanism of club cell CREB-dependent pathways contributing to mucin prosecretory effects of IL-1B. Dashed line represents hypothetical relationship that is supported by our data and the literature. IL-1B did not modify M3R and Rab3D, but loss of CREB did. Abbreviations: IL-1B, Interleukin 1β; VEH, vehicle; CREB1, cAMP responsive element binding protein 1; FOXA2, forkhead box A2; SPDEF, SAM pointed domain containing ETS transcription factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitated; NTC, no template control; gDNA, genomic DNA; Muc5ac, Mucin5ac; Muc5b, Mucin5b; Muc1, Mucin1; Muc4, Mucin4; Rab3D, RAS oncogene family; M3R, muscarinic receptor 3.
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