Osteoblastic Cells and the Hematopoietic Microenvironment: The Notch ligand Jagged1 Is Increased in Osteoblastic Stromal Cells by Parathyroid Hormone (PTH)Treatment.

Abstract

In the bone marrow, osteoblastic cells have recently been shown to be an important component of the hematopoietic stem cell (HSC) niche. We demonstrated that activation of the PTH Receptor (PTHR1) in osteoblastic cells results in expansion of HSC in vitro and in vivo. In addition, PTH treatment of mice undergoing myeloablative bone marrow transplantation using limiting numbers of donor cells dramatically improved their survival. Activation of the Notch signaling pathway was necessary to mediate the PTH-induced expansion of HSC (Calvi L.M., Adams G.B. et al., Nature 425, 841–846). This pathway, through cell-cell interactions plays a fundamental role in HSC self-renewal. Since normal primary murine stromal cells treated with PTH (1–34) had improved ability to support HSC, we analyzed the expression of the Notch ligand Jagged1 in these cells. Stromal cell Jagged1 has been shown to promote HSC self-renewal. Realtime PCR analysis of total RNA from primary stromal cells showed no changes in Jagged1 message in PTH compared to vehicle-treated cells. To assess whether this was due to rare and heterogeneous expression of Jagged1 in the stromal cell population, we performed immunocytochemical analysis of primary murine stromal cells treated with PTH(1–34). A small subpopulation of PTH-treated primary murine cells had a dramatic increase of Jagged 1 protein, while this subpopulation was not identified in vehicle-treated stromal cells. Since the PTHR1 is expressed in stromal osteoblastic cells, rat osteosarcoma UMR106 cells were chosen in order to study PTH-dependent Jagged1 expression in osteoblastic cells. A 10 fold time and dose-dependent increase in Jagged1 expression was measured by realtime PCR in RNA from UMR106 cells treated with PTH compared with vehicle. Western blot analysis using two distinct anti-Jagged1 antibodies confirmed the PTH-dependent increase in Jagged1 protein in UMR106 cells. In osteoblasts, PTH(1–34) is known to activate both the adenylate cyclase (AC) and the protein kinase C (PKC) signaling cascades downstream of the PTH1R. We independently determined the effect of activating either pathway on Jagged1. Forskolin, an AC activator, dramatically increased Jagged1 expression in a time and dose dependent fashion. Jagged1 protein was also increased by Forskolin. In contrast, Jagged1 protein levels were not increased by treatment with TPA, a PKC activator. In summary, osteoblastic cell treatment with PTH increases expression of the Notch ligand Jagged1 through activation of the AC signaling pathway downstream of the PTH1R. UMR106 provide a useful model to study the molecular mechanisms underlying osteoblastic PTH-dependent increases in Jagged1 levels, which are likely to play an important role in the PTH-induced enhanced osteoblastic support of HSC. Further definition of this pathway is likely to provide targets for pharmacologic manipulation of the HSC microenvironment.