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Characterization of in vitro cultured alveolar macrophages (AM) transcriptomes [bulk RNA-seq]

ABSTRACT: To identify molecular characteristics of WT AM without Wnt3a, and WT, b-catenin-deficient or HIF-1a-deficient AM with Wnt3a stimulation in vitro, we isolated RNA from AMs with various treatments and examined by bulk RNA-seq. We found a large number of gene profiles were altered following Wnt3a treatment in WT AMs, while b-catenin and HIF-1a deficiency largely abolished the effects of Wnt3a treatment on AM transcription, suggesting that b-catenin and HIF-1a mediates most downstream effects of Wnt3a in AMs. Indeed,HIF-1a-deficient AMs essentially phenocopied those of b-catenin-deficient AMs, with more than 80% of differentially expressed genes in HIF-1a-deficient AMs overlapping with b-catenin-deficient AMs following Wnt3a treatment.

ORGANISM(S): Mus musculus

PROVIDER: GSE164791 | GEO | 2021/05/05

REPOSITORIES: GEO

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Project description:HIF-1a activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HET HIF-1a fracture calluses are larger, stronger and stiffer than WT HIF-1a calluses due to decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. Introduction: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia following fracture activates hypoxia-inducible factor 1 alpha (HIF-1a) leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1a is a key regulator of bone regeneration Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1a deficiency (HET HIF-1a) and wild type littermates (WT HIF-1a). Fracture calluses and intact contralateral femurs from post fracture day (PFD) 21 and 28 (N=5-10) were subjected to MicroCT evaluation and 4-point bending in order to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10 and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. Results: Surprisingly, fracture calluses from HET HIF-1a mice exhibit greater mineralization and are larger, stronger and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, demonstrating higher expression of pro-apoptotic PP2A and lower expression of anti-apoptotic BCL2 in WT HIF-1a calluses. Subsequent TUNEL staining demonstrates that WT HIF-1a calluses contain larger numbers of TUNEL positive chondrocytes and osteoblasts than HET HIF-1a calluses. Conclusions: We conclude that partial HIF-1a deficiency results in decreased chondrocytic and osteoblastic apoptosis; thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration. Keywords: Bone, Fracture, Apoptosis, Hypoxia, Microarray

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Characterization of in vitro cultured alveolar macrophages (AM) transcriptomes [bulk RNA-seq]

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