Differential effects of PTH (1-34), PTHrP (1-36) and abaloparatide on the murine osteoblast transcriptome

Abstract

Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been tested and/or utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change >1 and a false discovery rate <0.05. These data were analyzed and compiled into heat maps and smear/volcano plots for each peptide. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes were shared only among PTH (1-34) and PTHrP; and 83 genes were shared among all three peptides. Gene ontology analyses show biological processes are similar between the three peptides but have some pathway-specific differences. Further analysis of the data illuminated that the three peptides increased expression of Vitamin D receptor (Vdr), Phosphodiesterase 10a (Pde10a), Cbp/p300-interacting transactivator 1 (Cited1), Wnt family member 11 (Wnt11), and Secreted frizzled related protein 4 (Sfrp4) mRNAs similarly to Rankl expression, i.e., in a differential expression pattern. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), or ABL for 4 h prior to harvest. Additionally, we analyzed mRNA abundance of several genes of interest, including Wnt4, Wnt7, Dkk1, Kcnk10, Hdac4, Epha3, Tcf7, Fzd5, Pp2r2a, and Dvl3 based on genes and pathways chosen from ontology analyses to be significantly regulated by each peptide. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these three peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis.