Regulation of Mesenchymal Stem Cell Osteogenic Differentiation by Glucocorticoid-induced Leucine Zipper (GILZ)

Abstract

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor ␥-2 (Ppar␥2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppar␥2 and C/ebp␣ were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.Bone marrow-derived mesenchymal stem cells (MSCs) 3 are pluripotent and can give rise to several distinct cell lineages, such as osteoblasts, adipocytes, chondrocytes, myocytes, and even neurons under appropriate conditions (1-4). Increasing evidence has shown that adipocytes and osteoblasts are two major pathways and that the relationship between these two is reciprocal, i.e. when the adipogenic pathway is blocked, the MSCs enter the osteogenic pathway, and vice versa (5-9). Thus, balanced MSC osteoblast and adipocyte differentiation is critical for the maintenance of healthy bone and lean body composition, and understanding of the mechanisms by which this balance is modulated will have significant medical implications in stem cell-based therapies. We reported previously that a glucocorticoid (GC)-inducible protein, called GC-induced leucine zipper (GILZ) (10), can inhibit the transcription of a key adipogenic regulator, peroxisome proliferator-activated receptor ␥-2 (Ppar␥2), and blocks adipocyte differentiation of 3T3-L1 cells (11). Gilz is a new member of the leucine zipper protein family and belongs to the transforming growth factor ␤-stimulated clone-22 (Tsc-22) family of transcription factors (12, 13). Members of this family contain three distinct domains: an N terminus TSC box, a middle leucine zipper domain, and a C terminus proline-rich domain. GILZ has been shown to interact with and inhibit the activities of the key inflammatory signaling mediators 15). GILZ can also interact with the mitogen-activated protein kinase family member, Raf1, resulting in inhibition of Raf-1 phosphorylation and, subsequently, inh...

Long Glucocorticoid-induced Leucine Zipper (L-GILZ) Protein Interacts with Ras Protein Pathway and Contributes to Spermatogenesis Control

“…Previous studies showed that the shorter isoform GILZ can inhibit T cell activation/proliferation (22, 24, 26, 44 -46). GILZ has also been shown to control proliferation and differentiation in other cell types including adipocytes, osteoblasts, and myoblasts (28,47,48). Conservation of most of the GILZ functional domains in L-GILZ isoform suggests that L-GILZ can act as suppressor of cell growth and differentiation.…”

Mechanism of Glucocorticoid-Induced Osteoporosis: An Update

“…To further study this problem, a retrovirus-mediated GILZ overexpression system was established in bone marrow MSCs/osteoprogenitor cells. Studies carried out in this system showed that GILZ has potent pro-osteogenic activity as demonstrated by significantly increased alkaline phosphatase activity, enhanced mineralized bone nodule formation, and the expression of osteoblast-associated genes such as Runx2, type I collagen, alkaline phosphatase, and osteocalcin [100]. Furthermore, our recent studies have shown that overexpression of GILZ can antagonize the inhibitory effect of TNF- on MSC osteogenic differentiation [101].…”

Stromal Cell-Derived Factor-1β Potentiates Bone Morphogenetic Protein-2-Stimulated Osteoinduction of Genetically Engineered Bone Marrow-Derived Mesenchymal Stem CellsIn Vitro

“…BMSCs derived from 18-month-old mice were subjected to retroviral-mediated transduction with DU3-GFP plasmid DNA, constructed in the replication defective DU3nlsLacZ vector by inserting the full-length coding region of Gfp cDNA. 29 BMSCs were then retransplanted in tibiae of 2-month-old C57BL/6J mice and their osteogenic potential was confirmed in vivo (unpublished data). After 4 weeks, animals were euthanized, the marrow cells harvested, and seeded at low density for clonal selection.…”

“…The isolation process, negative immunodepletion (CD11b, CD45R/B220, CD11c, plasmacytoid dendritic cell antigen-1 [PDCA-1]), positive immunoselection (stem cell antigen-1 [Sca-1]), and retroviral infection (GFP) have been described previously. 28,29 First, six mice per age group were euthanized by CO 2 overdose followed by thoracotomy. The femora and tibiae were dissected free of soft tissues and kept in cold PBS on ice.…”