Poly ADP-ribosylation (PARylation) is a post-translational modification catalyzed by poly (ADP-ribose) polymerase (PARP) family proteins such as PARP1. Although PARylation regulates important biological phenomena such as DNA repair, chromatin regulation, and cell death, little is known about the relationship between osteoblast differentiation and the PARylation cycle involving PARP1 and the poly (ADP-ribose)-degrading enzyme poly (ADP-ribose) glycohydrolase (PARG). Here, we examined the effects of PARP inhibitor olaparib, an approved anti-cancer agent, and PARG inhibitor PDD00017273 on osteoblast differentiation. Olaparib decreased alkaline phosphatase (ALP) activity and suppressed mineralized nodule formation evaluated by Alizarin Red S staining in preosteoblastic MC3T3-E1 cells, while PDD00017273 promoted ALP activity and mineralization. Furthermore, PDD00017273 up-regulated the mRNA expression levels of osteocalcin and bone sialoprotein, as osteoblast differentiation markers, and osterix as transcription inducers for osteoblast differentiation, whereas olaparib down-regulated the expression of these genes. These findings suggest that PARG inhibition by PDD00017273 accelerates osteoblast differentiation in MC3T3-E1 cells. Thus, PARG inhibitor administration could provide therapeutic benefits for metabolic bone diseases such as osteoporosis.

The parathyroid gland plays an essential role in mineral and bone metabolism. A cultivation for physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. Here, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. The parathyroid cell differentiation occurred in accordance with embryologic development. The differentiated cells, which expressed parathyroid hormone, adopted a unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

PARP inhibitors have been developed as anti-cancer agents based on synthetic lethality in homologous recombination deficient cancer cells. However, resistance to PARP inhibitors such as olaparib remains a problem in clinical use, and the mechanisms of resistance are not fully understood. To investigate mechanisms of PARP inhibitor resistance, we established a BRCA1 knockout clone derived from the pancreatic cancer MIA PaCa-2 cells, which we termed C1 cells, and subsequently isolated an olaparib-resistant C1/OLA cells. We then performed RNA-sequencing and pathway analysis on olaparib-treated C1 and C1/OLA cells. Our results revealed activation of cell signaling pathway related to NAD⁺ metabolism in the olaparib-resistant C1/OLA cells, with increased expression of genes encoding the NAD⁺ biosynthetic enzymes NAMPT and NMNAT2. Moreover, intracellular NAD⁺ levels were significantly higher in C1/OLA cells than in the non-olaparib-resistant C1 cells. Upregulation of intracellular NAD⁺ levels by the addition of nicotinamide also induced resistance to olaparib and talazoparib in C1 cells. Taken together, our findings suggest that upregulation of intracellular NAD⁺ is one of the factors underlying the acquisition of PARP inhibitor resistance.