c-Myc regulates self-renewal in bronchoalveolar stem cells

Abstract

Background: Bronchoalveolar stem cells (BASCs) located in the bronchoalveolar duct junction are thought to regenerate both bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury. The mechanisms directing self-renewal in BASCs are poorly understood.

Methods: BASCs (Sca-1(+), CD34(+), CD31(-) and, CD45(-)) were isolated from adult mouse lung using FACS, and their capacity for self-renewal and differentiation were demonstrated by immunostaining. A transcription factor network of 53 genes required for pluripotency in embryonic stem cells was assessed in BASCs, Kras-initiated lung tumor tissue, and lung organogenesis by real-time PCR. c-Myc was knocked down in BASCs by infection with c-Myc shRNA lentivirus. Comprehensive miRNA and mRNA profiling for BASCs was performed, and significant miRNAs and mRNAs potentially regulated by c-Myc were identified. We explored a c-Myc regulatory network in BASCs using a number of statistical and computational approaches through two different strategies; 1) c-Myc/Max binding sites within individual gene promoters, and 2) miRNA-regulated target genes.

Results: c-Myc expression was upregulated in BASCs and downregulated over the time course of lung organogenesis in vivo. The depletion of c-Myc in BASCs resulted in decreased proliferation and cell death. Multiple mRNAs and miRNAs were dynamically regulated in c-Myc depleted BASCs. Among a total of 250 dynamically regulated genes in c-Myc depleted BASCs, 57 genes were identified as potential targets of miRNAs through miRBase and TargetScan-based computational mapping. A further 88 genes were identified as potential downstream targets through their c-Myc binding motif.

Conclusion: c-Myc plays a critical role in maintaining the self-renewal capacity of lung bronchoalveolar stem cells through a combination of miRNA and transcription factor regulatory networks.

Figure 1. Characterization of BASCs.

(A) Methodology of FACS sorting of BASC. First, cells were gated on the CD31⁻ CD45⁻ population, and then subsequently gated on the CD31⁻ CD45⁻ CD34⁺ ScaI⁺ population (BASCs). (B) Phase-contrast images of clonal growth of BASCs on day 6 and BASCs in culture after several generations in serial culture (200×). (C) Identification of BASCs by dual-color immunofluorescent staining for anti-SP-C (red) and anti-CCA (green). (D) Differentiation of BASCs in SAEM media was identified by staining for anti-SP-C (red), anti-CCA (green) and anti-Aquaporin (green).

Figure 2. Expression of stem cell transcription factors.

(A) In BASCs. (B) In mouse lung in different developmental stages. (C) In K-ras mutant activated lung tumor tissue. In total, expression of 53 stem cell transcription factors were measured including 34 genes from a transcription network for pluripotency of embryonic stem cells , and 19 genes from candidates for reprogramming fibroblasts into a pluripotent embryonic stem cell-like state using quantitative real-time PCR. β-actin was used as an internal control. Statistically significant differences are shown. Values are means ± SD of the fold increase compared with adult mouse lung (n = 3).

Figure 3. Inhibition of c-Myc expression in BASCs.

(A) BASCs were plated in 24-well plates at 40,000 cells per well and incubated in complete medium overnight to achieve 50% confluence. BASCs were infected with viral supernatant including control shRNA or two different c-Myc shRNAs. At day 3, day 5 and day 7 after infection, the cells were washed with PBS and images were taken under both normal light and blue light for GFP expression. (B) BASCs were suspended and stained with trypan blue at the indicated time points. Stained cells were placed in a hemocytometer and viable (unstained) cells were counted. Plotted values are the means ± SD of three replicates. (C) After several generations of puromycin selection, stable c-Myc knock down cell lines were established. c-Myc mRNA and protein expression were measured by real-time PCR and western blotting and cells were counted at the indicated time points.

Figure 4. c-Myc-regulated mRNA expression pattern and gene ontology analysis.

(A) Heat map of significantly changed genes in BASCs with and without c-Myc. (B). Analysis of enrichment of GO biological process categories for each expression pattern with p<0.05 and fold enrichment >2 are listed (ranked by count number).

Figure 5. Expression patterns of c-Myc-regulated miRNA and miRNA targets.

(A) Heat map of significantly changed miRNAs in BASCs with and without c-Myc. (B) Targeted genes and the genes numbers for each individual miRNA identified through overlapping the potential targets from the miRBase and TargetScan predicted mRNA targets (blue). (C) Effect of c-Myc on the expression of miR-34 family. (D) Relative expression of mRNA targeted by miR-34-3p using real-time PCR (n = 3, mean ± SE).

Figure 6. Schematic representation of c-Myc regulating cell proliferation and controlling cell fate in BASCs through both transcriptional and post-transcriptional pathways.

The depletion of c-Myc in BASCs resulted in decreased proliferation and cell death. 250 mRNAs and 17 miRNAs were dynamically regulated in c-Myc depleted BASCs. The model (from our work and others) depicts c-Myc regulating downstream gene expression through two distinct mechanisms. 1) Transcriptional level: c-Myc directly regulates mRNA transcription with its partner Max through c-Myc/Max binding sites in the 5′-flanking promoter region of target genes. 88 genes were identified as potential downstream genes through a position weight matrix based method in conjunction with changes in mRNA level. 2) Post-transcriptional level: c-Myc indirectly regulates mRNA and protein expression through miRNAs. Myc can regulate miRNA expression through c-Myc/Max binding sites , and we identified Myc-regulated miRNAs by comparing the c-Myc depleted group with controls. The mature miRNA can then negatively regulate gene expression through one of 2 mechanisms; a) by miRNA/mRNA binding and degradation, which is dependent on sequence complementarity between the miRNA and the target mRNA (57 genes were identified as targets of miRNAs through miRBase and TargetScan-based computational mapping); or b) miRNAs suppressing gene expression by blocking protein translation independent of changes in mRNA level , .