Kurt D. Hankenson

Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ

Mesenchymal precursor cells have the potential to differentiate into several cell types, including adipocytes and osteoblasts. Activation of Wnt/β-catenin signaling shifts mesenchymal cell fate toward osteoblastogenesis at the expense of adipogenesis; however, molecular mechanisms by which Wnt signaling alters mesenchymal cell fate have not been fully investigated. Our prior work indicates that multipotent precursors express adipogenic and osteoblastogenic transcription factors at physiological levels and that ectopic expression of Wnt10b in bipotential ST2 cells suppresses expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and increases expression of Runx2, Dlx5, and osterix. Here, we demonstrate that transient activation of Wnt/β-catenin signaling rapidly suppresses C/EBPα and PPARγ, followed by activation of osteoblastogenic transcription factors. Enforced expression of C/EBPα or PPARγ partially rescues lipid accumulation and decreases mineralization in ST2 cells expressing Wnt10b, suggesting that suppression of C/EBPα and PPARγ is required for Wnt/β-catenin to alter cell fate. Furthermore, knocking down expression of C/EBPα, PPARγ, or both greatly reduces adipogenic potential and causes spontaneous osteoblastogenesis in ST2 cells and mouse embryonic fibroblasts, suggesting that Wnt signaling alters the fate of mesenchymal precursor cells primarily by suppressing C/EBPα and PPARγ.

Integration of BMP, Wnt, and Notch signaling pathways in osteoblast differentiation

Bone marrow‐derived mesenchymal stem cells (MSCs) are multipotent progenitors that can commit to osteoblast, chondrocyte, adipocyte, and several other lineages. The proper utilization of stem cells for clinical applications requires an integrated understanding of multiple signal inputs that control maintenance of stemness, proliferation, commitment, and differentiation. Various signaling pathways have been implicated in the regulation of MSC differentiation; however, complexities of pathway interactions, as well as seemingly contradictory results in the literature, create an often confusing and disjointed knowledge base. Several recent publications explore the integration of signaling pathways such as BMP, Wnt, Notch, Hedgehog, and Fibroblast Growth Factors in MSC osteoblast differentiation. The transcription factor Cbfa1/Runx2 has been implicated in these pathways as a potential focal point for signaling integration. This review will outline the current understanding of these pathways and indicate where both spatiotemporal effects during differentiation and comparable experimental conditions need to be considered in order to clarify the outcome(s) of differing regulatory levels of these signaling pathways. J. Cell. Biochem. 112: 3491–3501, 2011.

Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6.

Bone marrow‐derived mesenchymal stem cells (MSC) are multipotent, self‐renewing, mesodermal‐origin stem cells that are sequestered in the endosteal compartment. MSC are maintained in a relative state of quiescence in vivo but in response to a variety of physiological and pathological stimuli, proliferate and differentiate into osteoblasts, chondrocytes, adipocytes, or hematopoiesis‐supporting stromal cells. Little is understood regarding the cellular or molecular events underlying MSC fate decisions. We report that human MSC (hMSC) cultured in defined, serum‐free conditions respond to a narrow spectrum of growth factors with osteogenic commitment, differentiation, and hydroxyapatite deposition. Of the osteogenic factors we examined, only treatment with bone morphogenetic protein (BMP) results in osteoinduction under defined serum‐free conditions. Among BMP‐2, 4, 6, and 7, BMP‐6 is the most consistent and potent regulator of osteoblast differentiation and, of these BMPs, only BMP‐6 gene expression is detected prior to hMSC osteoblast differentiation. Addition of exogenous BMP‐6 to hMSC induces the expression or upregulation of a repertoire of osteoblast‐related genes including type I collagen, osteocalcin, bone sialoprotein, and their regulatory transcription factors Cbfa1/Runx2, and Osterix. This translates into increased production of osteogenic extracellular matrix (ECM) with subsequent hydroxyapatite deposition. J. Cell. Biochem. 98: 538–554, 2006.

Angiogenesis in Bone Regeneration

Angiogenesis is a key component of bone repair. New blood vessels bring oxygen and nutrients to the highly metabolically active regenerating callus and serve as a route for inflammatory cells and cartilage and bone precursor cells to reach the injury site. Angiogenesis is regulated by a variety of growth factors, notably vascular endothelial growth factor (VEGF), which are produced by inflammatory cells and stromal cells to induce blood vessel in-growth. A variety of studies with transgenic and gene-targeted mice have demonstrated the importance of angiogenesis in fracture healing, and have provided insights into regulatory processes governing fracture angiogenesis. Indeed, in animal models enhancing angiogenesis promotes bone regeneration, suggesting that modifying fracture vascularization could be a viable therapeutic approach for accelerated/improved bone regeneration clinically.

Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell–matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.

Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.

The phenotype of thrombospondin 2 (TSP2)–null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2‐null mice. Histomorphometric analysis showed that the mid‐diaphyseal endosteal bone formation rate (BFR) of TSP2‐null mice was increased in comparison with that of wild‐type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2‐null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2‐null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2‐null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased. (J Bone Miner Res 2000;15:851–862)

Pleiotropic Phenotype of a Genomic Knock-In of an RGS-Insensitive G184S Gnai2 Allele

ABSTRACT Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Gαi2 subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Gαi2G184S knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Gαi2+/G184S mice show some but not all of these abnormalities. Thus, loss of RGS actions at Gαi2 produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.

Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Wnt11 signals through both canonical (β-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases β-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/β-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through β-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.

Diminished Callus Size and Cartilage Synthesis in α1β1 Integrin-Deficient Mice during Bone Fracture Healing

Integrins mediate cell adhesion to extracellular matrix components. Integrin α1β1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in α1 integrin (α1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in α1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in α1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the α1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and α1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of α1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that α1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Leptin functions peripherally to regulate differentiation of mesenchymal progenitor cells.

Leptin functions through a well‐documented central neuroendocrine pathway to regulate bone mass. However, the ability of leptin to modulate bone mass through a peripheral mechanism has been debated due to conflicting in vitro results and lack of sufficient in vivo models. We utilized mice with LoxP sites introduced into the long‐form leptin receptor (ObRb) gene to determine how leptin regulates mesenchymal progenitor cell (MPC) differentiation and osteoblast function in vitro and in vivo. Rapid phosphorylation of Stat3 after leptin treatment of bone marrow stromal cells (BMSCs) from mice with conditional deletion of ObRb in macrophages (LysMCre+F/F) confirmed expression of functional leptin receptors by BMSCs. Adenovirus‐Cre mediated disruption of ObRb in primary stromal cells decreased mineralization and increased adipogenesis. In contrast, BMSCs harvested from leptin‐signaling deficient Ob/Ob or Db/Db mice showed increased mineralization. To determine the physiologic relevance of these differences, mice with cell‐specific deletion of ObRb in mesenchymal precursors (3.6Cre+F/F) or osteoblasts (2.3Cre+F/F) were generated. Although the 2.3Cre+F/F mice were grossly normal, the 3.6Cre+F/F mice displayed mild obesity that was not attributed to food intake. Femurs of 3.6Cre+F/F animals showed a 58%–61.9% increase in trabecular bone volume and a 65.5%–74% increase in bone mineral density. Cortical volume and mineral content were also increased 18%–22%. Primary 3.6Cre+F/F BMSCs recapitulated the high mineralization phenotype of Ob/Ob and Db/Db BMSCs. We conclude that leptin may have multiple peripheral roles depending on the differentiation state of MPC. Leptin (a) helps maintain MPCs in an undifferentiated state and (b) promotes mineralization of more differentiated osteoblasts. STEM Cells 2010;28:1071–1080