Maine Medical Center, Medical Education, Maine Medical Center Research Institute
Parathyroid hormone (PTH) analogs are the main anabolic pharmacological agent for osteoporosis. PTH is an endogenous hormone. Its amino acids 1-34 bind the parathyroid hormone receptor (PTH1r), a G-coupled protein receptor expressed in kidney, fat, and bone. PTH increases trabecular bone mass by promoting the differentiation of the mesenchymal stem cell (MSC) into the osteogenic lineage. The Zinc Finger Protein 467 (Zfp467) is a potential downstream target of PTH1r and an important mediator of the MSC into the adipogenic lineage. Taken together, we ask whether Zfp467 knockout cells will show greater osteogenic potential and increased sensitivity to PTH treatment. We also seek to investigate a mechanistic signaling pathway of PTH1r involving Zfp467.
Calvarial osteoblast (COB) and bone marrow stromal cells (BMSCs) from Zfp467 wild type (WT) and knockout (KO) mice were osteogenically differentiated and treated with either continuous (48h) or intermittent 6h/42h) PTH for 7-14 days. At 7 and 14 days, alkaline phosphatase (ALP) and alizarin red staining were conducted, respectively. At 7 days, qPCR was used to analyze genes involved in osteogenesis, adipogenesis, WNT signaling, and mitochondrial respiration. ELISA was used to measure cAMP levels. Seahorse XF96 assays were used to measure oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs). Western blot was used to measure PTH1r. Additionally, adipogenic differentiation and Oil Red O staining were performed on BMSCs.
ALP and alizarin red results showed that Zfp467 KO cells exhibited increased osteogenesis and an increased response to PTH treatment (continuous and intermittent) as compared to WT controls. qPCR analysis of Alp, RankL, and Sp7 further supported an increased osteogenic potential of the KO. Also, Oil Red O staining revealed suppressed adipogenesis in KO BMSCs; qPCR analysis showed suppressed Adiponectin and Ppary in KO COBs. Additionally, Pth1r and PTH1r expressions were significantly higher in KO; short PTH treatments (~10m) induced a remarkable reduction in Zfp467 of WT cells. Furthermore, the KO showed suppressed Pgc1a, similar OCR, and increased ECAR as compared to WT. The KO also exhibited higher cAMP levels and was more responsive to PTH-induced increases of cAMP after 10 minutes of PTH exposure. However, qPCR analysis of Lef1 and Sost showed no difference regarding the WNT pathway.
Our data support an anti-osteogenic and pro-adipogenic role for Zfp467. The KO displays less adipogenesis, more osteogenesis, and is consistently more sensitive to the osteogenic effects of PTH. The upregulation of Pth1r and PTH1r in KO cells offers an explanation for this increased sensitivity. We propose a mechanism where the suppression of Zfp467 upregulates Pth1r and PTH1r activation suppresses Zfp467, resulting in a constitutively active positive feedback loop. Further still, the KO shows potentially suppressed mitochondrial biogenesis (Pgc1a), similar oxidative phosphorylation (OCR), increased glycolysis (ECAR), and increased PKA signaling (cAMP), yet their exact connections to the PTH1r-ZFP467 signaling pathway have yet to be investigated.
Liu, H; Calle, I Leon; Le, P T.; Baron, R; and Rosen, C J., "Role of Zfp467in Mediating the Anti-Adipogenic and Pro-Osteogenic Effects of PTH: An In-VitroAnalysis" (2020). Costas T. Lambrew Research Retreat 2020. 34.