Osteoblasts Directly Control Lineage Commitment of Mesenchymal Progenitor Cells through Wnt Signaling

Yu Zheng,Hong Zhou,Markus J. Seibel,Colin R. Dunstan,Wendy Mak

doi:10.1074/jbc.m702687200

Abstract

Lineage commitment of mesenchymal progenitor cells is still poorly understood. Here we demonstrate that Wnt signaling by osteoblasts is essential for mesenchymal progenitor cells to differentiate away from a default adipogenic into an osteoblastic lineage. Dominant adipogenesis and reduced osteoblastogenesis were observed in calvarial cell cultures from transgenic mice characterized by osteoblast-targeted disruption of glucocorticoid signaling. This phenotypic shift in mesenchymal progenitor cell commitment was associated with reciprocal regulation of early adipogenic and osteoblastogenic transcription factors and with a reduction in Wnt7b and Wnt10b mRNA and beta-catenin protein levels in transgenic versus non-transgenic cultures. Transwell co-culture of transgenic mesenchymal progenitor cells with wild type osteoblasts restored commitment to the osteoblast lineage. This effect was blocked by adding sFRP1, a Wnt inhibitor, to the co-culture. Treatment of transgenic cultures with Wnt3a resulted in stimulation of osteoblastogenesis and suppression of adipogenesis. Our findings suggest a novel cellular mechanism in bone cell biology in which osteoblasts exert direct control over the lineage commitment of their mesenchymal progenitor through Wnt signaling. This glucocorticoid-dependent forward control function indicates a central role for osteoblasts in the regulation of early osteoblastogenesis.

Highlights

Glucocorticoid (GC) signaling through its cognate receptor is known to influence osteoblast and adipocyte lineage commitment both in vitro and in vivo and is, likely to have a role in regulating cellular interactions driving cell commitment
Osteoblast Differentiation Is Inhibited, and Adipocyte Differentiation Is Increased in Col2.3–11␤HSD type 2 (11␤HSD2) tg Culture—Primary osteoblast cultures were generated from the calvaria of 1-dayold Col2.3–11␤HSD2 tg mice and wild type (WT) littermates and grown under osteogenic conditions
In light of the fact that the transgene is exclusively expressed in mature osteoblasts [7, 11], these results indicate that early precursor cells derived from transgenic, 11␤HSD2-overexpressing animals experience a major shift in lineage commitment from osteoblast to adipocyte

Summary

EXPERIMENTAL PROCEDURES

Transgenic Mice—Col2.3–11␤HSD2 transgenic mice were generated as described previously [11] and were provided as a gift by Dr Barbara Kream (Dept. of Medicine, University of Connecticut Health Center, Farmington, CT). Cells collected from the first digestion, designated population 1 (P1), were cultured in 24-well plates. Cells collected from third and fourth digestions were pooled and designated as populations 3 and 4 (P3– 4) This population or cells was cultured in transwell inserts (pore size 3 ␮m, BD Biosciences) overnight and were transferred in the inserts to the 24-well plates containing P1 cells. RT-PCR and Real-time RT-PCR—Total RNA was isolated from primary mouse calvaria cell cultures of populations P1 and P2– 4 using NucleoSpin (Machery-Nagel, Easton, PA) according to the manufacturer’s instructions. The remaining pellet containing the nuclear fraction was spun for an additional 15 min at 15,000 ϫ g, washed with lysis buffer, and resuspended in sucrose buffer (250 mM sucrose, 20 mM Tris, pH 7.4, and protease inhibitor) Both cellular and nuclear proteins were separated by SDS-PAGE (7.5% polyacrylamide) and transferred onto nitrocellulose filters. Mouse-specific primer pairs used for RT-PCR and real-time RT-PCR SGK, serum and glucocorticoid-induced kinase; BSP, bone sialoprotein; ALP, alkaline phosphatase

Forward Primer

Reverse primer

RESULTS

DISCUSSION