Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression

Abstract

Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines.

Suppression of ornithine decarboxylase promotes osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells

“…1). By analyzing both the catabolic and anabolic enzyme expression of polyamine biosynthesis, it is suggested that in the presence of exogenous polyamines, ODC was suppressed and SSAT was stimulated to prevent rapid increase in intracellular polyamines [24]. Conversely, SSAT was suppressed when hBMSCs were treated with DFMO or siODC possibly to prevent further depletion of the polyamine pool (Fig.…”

“…Primer sequences and conditions for real-time PCR analysis are the same as those reported previously [24].…”

“…Cells were seeded at 3000 cells/well in 96-well plates for cell viability assay and alkaline phosphatase (ALP) activity assay, at 10 4 cells/well in 24-well plates for alizarin red S and oil red O staining, at 5 Â 10 4 cells/well in 6-well plates for total RNA and protein extraction, and at 5000 cells/cm 2 in 10-cm dishes for HPLC sample preparation. Alizarin red S and oil red O staining were performed as previously described [24].…”

“…In our previous report, the mRNA level of ODC was reduced in the presence of exogenous polyamines such as putrescine, spermidine and spermine, which enhance the osteogenic differentiation of human bone marrow-derived stem cells (hBMSCs) [24]. It is therefore hypothesized that suppression of ODC and thus depletion of endogenous polyamines may be correlated with the osteogenic induction of hBMSCs.…”

Preparation of Antispermidine/Spermine-N1-Acetyltransferase Monoclonal Antibodies

“…The enhanced SSAT activity affects osteoblastogenesis and seemed to be mediated through mechanisms other than polyamine depletion. (10) Here, monoclonal antibodies (mAbs) against SSAT were prepared to understand the molecular mechanisms underlying the complex roles of SSAT, and to find additional biological functions of this protein, as well as to investigate its mechanisms of action and how its levels of expression are regulated.…”