Transcriptional profiling of PRKG2-null growth plate identifies putative down-stream targets of PRKG2

Abstract

Background: Kinase activity of cGMP-dependent, type II, protein kinase (PRKG2) is required for the proliferative to hypertrophic transition of growth plate chondrocytes during endochondral ossification. Loss of PRKG2 function in rodent and bovine models results in dwarfism. The objective of this study was to identify pathways regulated or impacted by PRKG2 loss of function that may be responsible for disproportionate dwarfism at the molecular level.

Methods: Microarray technology was used to compare growth plate cartilage gene expression in dwarf versus unaffected Angus cattle to identify putative downstream targets of PRGK2.

Results: Pathway enrichment of 1284 transcripts (nominal p < 0.05) was used to identify candidate pathways consistent with the molecular phenotype of disproportionate dwarfism. Analysis with the DAVID pathway suite identified differentially expressed genes that clustered in the MHC, cytochrome B, WNT, and Muc1 pathways. A second analysis with pathway studio software identified differentially expressed genes in a host of pathways (e.g. CREB1, P21, CTNNB1, EGFR, EP300, JUN, P53, RHOA, and SRC). As a proof of concept, we validated the differential expression of five genes regulated by P53, including CEBPA, BRCA1, BUB1, CD58, and VDR by real-time PCR (p < 0.05).

Conclusions: Known and novel targets of PRKG2 were identified as enriched pathways in this study. This study indicates that loss of PRKG2 function results in differential expression of P53 regulated genes as well as additional pathways consistent with increased proliferation and apoptosis in the growth plate due to achondroplastic dwarfism.

Figure 1

Enriched genes in PRKG2-null growth plate in the P53 pathway. The P53 differentially expressed gene pathway as drawn by pathway studio software. A total of 38 target genes were identified, many of which were also identified as over-enriched for their own downstream targets by Pathway Studio. Additional details regarding the associated targets and publications supporting these target predictions are provided in Additional file 3.

Figure 2

Validation of DE genes in the P53 pathway. The P53 pathway genes validated as differentially expressed by quantitative real-time PCR (p < 0.05). Transcripts BRCA1, BUB1, CEBPA, and CD58 were found to be differentially expressed using the ddCT method, while VDR was differentially expressed when comparing the natural log (ln) starting transcript copy number between genotypes. Fold changes in gene expression are presented, where the genotype with the lowest expression level was set to one to facilitate visualization of fold change differences.