Targeting intercellular signals for bone regeneration from bone

[Cell Cycle 7:14, 2106-2111; 15 July 2008]; ©2008 Landes Bioscience

Perspective

Targeting intercellular signals for bone regeneration from bone marrow mesenchymal progenitors Fanxin Long Department of Medicine; Department of Developmental Biology; Washington Universtiy Medical School; St. Louis, Missouri USA

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Introduction

lack of reliable molecular markers, although some researchers have operationally defined MSC as cells whose progenies, after in vitro expansion, can induce ectopic formation of both bone and marrow (as opposed to bone only) in a mouse transplantation model,3 and have shown that selection for cell-surface CD146 (MCAM) expression enriched MSC in preparations of human bone marrow stromal cells (BMSC).4 Nonetheless, both MSC and the progenitors (herein collectively termed bone marrow mesenchymal progenitors) can be detected in the clonogenic subset of adherent bone marrow stromal cells (BMSC), known as colony-forming unit-fibroblastic (CFUF).5-8 Thus, the size of the mesenchymal progenitor pool in a bone marrow sample can be estimated from CFU-F frequencies among BMSC. Reduced bone formation is a major cause for bone loss during aging and in osteoporosis patients.9,10 Because osteoblast differentiation is thought to be a rate-limiting step for bone formation,11 decreased osteoblastogenesis likely contributes to age- and osteoporosis-related bone loss. The cause for the reduced osteoblast production however, is not well understood. While some researchers have proposed agerelated deterioration of the mesenchymal progenitors as a potential reason,12-14 others have suggested other mechanisms.15,16 One such alternative mechanism could involve intercellular signals that direct fates of the progenitors. Indeed, intercellular signals within the bone marrow microenvironment may constitute a “molecular niche” to control fate decisions of the mesenchymal progenitors; unfavorable changes to the niche are expected to cause a decrease in osteoblastogenesis. Regardless of the mechanism, the sustained presence of mesenchymal progenitors in the bone marrow of aged or osteoporotic humans has raised the possibility that pharmaceutical enhancement of osteogenic signals in the bone marrow environment may represent an effective “niche therapy” in promoting bone formation in vivo. In this article I highlight some of the intercellular signals that have been implicated in osteoblastogenesis in the mouse.

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There are tremendous unmet clinical needs for effective strategies to enhance bone regeneration in vivo. The sustained presence of multipotent mesenchymal progenitors in the bone marrow in aged and osteoporotic individuals offers the potential for therapeutic interventions to induce osteoblast production from the resident progenitors. Recent advances in understanding the intercellular signals governing osteogenic decisions may provide targets for developing novel bone-enhancing therapeutics.

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Key words: mesenchymal stem cells, osteoblast, osteoclast, osteoporosis, bone regeneration, notch, Wnt, Hh, Bmp, Fgf

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The adult human skeleton is a dynamic organ undergoing continuous remodeling. Bone homeostasis is achieved through balancing bone formation by osteoblasts and bone resorption by osteoclasts. On the other hand, excessive resorption over formation leads to osteoporosis characterized by low bone mass and bones prone to fractures. The National Organization for Osteoporosis estimates that one in two women and one in four men over 50 will eventually suffer an osteoporosis-related fracture. Currently, the bisphosphonate class of antiresorptive drugs that inhibit osteoclast activity is the mainstay of osteoporosis treatment; these drugs, however, reduce bone remodeling and do not restore normal microarchitecture to osteoporotic bones.1 Although Teriparatide based on human parathyroid hormone is designed to stimulate bone formation, its clinical approval is limited to severe osteoporosis patients and to relatively short-term treatment. Thus, additional means that enhances new bone formation and improves bone quality is clearly critical to effective treatment of osteoporosis. The adult bone marrow contains multipotential mesenchymal progenitors that, in their native environment, collectively produce adipocytes, osteoblasts and the hematopoiesis-supporting stromal cells. These primitive progenitors are often referred to as mesenchymal stem cells (MSC) in the literature, but they likely include both bona fide stem cells and the more committed progenitors.2 A clear separation of MSC from the progenitors is difficult at present due to the Correspondence to: Fanxin Long; Department of Medicine; Department of Developmental Biology; Washington Universtiy Medical School; St. Louis, Missouri 63110 USA; Tel.: 314.454.8795; Email: [email protected] Submitted: 05/02/08; Accepted: 05/08/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6257 2106

Notch Signaling Inhibits Osteoblastogenesis Notch signaling mediates communication between neighboring cells to control cell fate decisions both during embryogenesis and in postnatal life. In the canonical Notch pathway, the single-pass transmembrane cell surface Notch receptors (Notch1-4 in mammals) undergo proteolytic cleavages upon binding of ligands (Jagged1, 2 and Delta-like 1, 3, 4 in mammals, also single-pass transmembrane proteins) presented on a neighboring cell surface.17 As a result, the

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Intercellular signals for osteoblastogenesis

Figure 1. Multiple intercellular signals controlling osteoblastogenesis and bone homeostasis. See text for details. ↑: stimulation; ⊥: inhibition; MSC: mesenchymal stem cells; OB: mature osteoblasts; HSC: hematopoietic stem cells; MC: macrophage progenitor cells; OC: osteoclasts.

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The discovery that Notch normally functions as a brake for osteoblastogenesis from bone marrow mesenchymal progenitors raises the possibility that pharmacological inhibition of the pathway may stimulate osteoblast production much needed in osteoporotic patients. On the other hand, both our studies and those by others also demonstrated that sustained Notch inhibition also enhanced osteoclastogenesis. Thus, long-term Notch inhibition is unlikely to achieve the desired anabolic effect in bone. However, short pulses of Notch inhibition may enhance osteoblastogenesis, without inducing overproduction of osteoclasts. Alternatively, treatments combining Notch inhibition with anti-resorptive therapies may increase bone mass.

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Notch intracellular domain (NICD) is released from the plasma membrane and translocates to the nucleus where it interacts with a transcription factor of the CSL family (RBP-Jκ/CBF-1 in mammals) to activate transcription of target genes including those of the Hes/ Hey family, which are themselves transcription factors containing a conserved basic helix-loop-helix (bHLH) domain.18 The intramembrane cleavage and release of NICD requires a functional γ-secretase complex which contains either presenilin 1 (PS1) or 2 (PS2) as the catalytic subunit.19-21 To investigate the potential role of Notch signaling in osteoblastogenesis, we recently genetically removed critical components of the Notch pathway in the developing skeleton.22 We find that removal of either PS1 and PS2, or Notch1 and Notch2 in the limb mesenchyme did not overtly affect skeletal morphogenesis in the embryo, but markedly enhanced trabecular bone mass in adolescent mice. The high-bone-mass phenotype was present in the face of increased osteoclastogenesis and total resorption activities in the Notch-deficient mice. Surprisingly, when subjected to CFU-F and in vitro differentiation assays, the bone marrow of the high-bonemass animals was found to possess substantially fewer mesenchymal progenitors than their normal littermates. Consequently, the deficit in mesenchymal progenitor numbers, in combination with the increased osteoclast production, caused the Notch-deficient mice that began with a high bone mass at a young age, to rapidly develop severe osteopenia when they aged. Mechanistically, we find that Notch inhibits osteoblast differentiation in progenitors by inducing Hey1 and HeyL that in turn diminish Runx2 transcriptional activity via physical interaction. Additionally, Notch in the more mature osteoblast-lineage cells functions to control osteoclastogenesis by inhibiting expression of the pro-osteoclastogenic Rankl and stimulating that of the anti-osteoclastogenic Opg. The indirect regulation of osteoclast production by Notch signaling in osteoblasts has also been demonstrated by two additional studies.23,24 Overall, these studies establish Notch signaling in osteoblast-lineage cells as a critical mechanism for regulating bone homeostasis in mice: it not only directly controls osteoblastogenesis in the progenitors, but also acts in the more mature cells to regulate osteoclastogenesis indirectly (Fig. 1). Although our CFU-F assays indicated that the population of MSC (a subset of the CFU-F-producing cells) was likely reduced in the Notch-deficient mice, these results did not distinguish whether the deficiency was directly caused by loss of Notch in MSC, or secondary to Notch-deficiency in the more committed progenitors. Definitive demonstration of a direct role of Notch in bona fide MSC will require definitive identification of these cells and direct manipulation of Notch activity in that population.

Wnt Signaling Promotes Bone Formation

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Contrary to Notch, Wnt signaling has been shown to enhance bone formation. The Wnt family of glycoproteins plays essential roles both during development and in disease. Upon binding membrane receptors, Wnts activate intracellular pathways either requiring or independent of β-catenin, known as canonical or noncanonical signaling, respectively.25,26 In canonical Wnt signaling, Wnt binding to Frizzled receptors and the low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), activates the cytoplasmic signaling protein Dishevelled to stabilize cytosolic β-catenin; β-catenin, upon entering the nucleus, stimulates transcription of downstream target genes via lymphoid enhancer-binding factor-1 (Lef-1) and T cell factors (Tcf1, 3, 4). The mechanism responsible for the nuclear localization of β-catenin has been long elusive, but we have recently shown that Rac1 and JNK activation leading to specific phosphorylation of β-catenin is critical for this process.27 The intracellular cascades for noncanonical Wnt signaling are less well understood, but appear to involve phosphatidylinositol and Rho-family small GTPase signaling. The importance of canonical Wnt signaling in bone formation is supported by genetic evidence. In humans, loss- or gain-of function mutations in LRP-5 were linked with the osteoporosispseudoglioma syndrome,28 or a high bone density syndrome,29,30 respectively. Similarly, mice lacking Lrp-5,31 or Wnt10b32 showed less bone postnatally, and reduction of Lrp-6 was shown to further reduce bone mass in Lrp5-null mice.33 Conversely, mice lacking the Wnt antagonist secreted Frizzled related protein 1 developed more bone in postnatal life.34 While these results implicate canonical Wnt signaling in postnatal bone accrual, genetic deletion of β-catenin from early osteogenic progenitors has demonstrated that the pathway is necessary for osteoblast development in the mouse embryo.35-38 Interestingly, although β-catenin is required for the

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The bone morphogenetic proteins (BMPs), originally discovered based on their ability to induce ectopic cartilage and bone formation in animals, belong to the transforming growth factor-β (TGFβ) superfamily of secreted growth factors, and play critical roles in a wide variety of tissues during development.53 By binding as dimers to receptor complexes composed of heterotetramers of type I and II serine-threonine kinase receptors, BMP proteins activate a Smaddependent pathway to regulate gene expression of the downstream targets. In this mechanism, BMP binding triggers the type II receptor (BMPRII, ActRII or ActRIIB) to phosphorylate the type I receptor (ALK2/ActR1, ALK3/BMPRIA or ALK6/BMPRIB), which in turn activates the receptor Smads (R-Smads: Smad1, 5 and 8) through site-specific phosphorylation. The phosphorylated R-Smads then form a complex with the common partner Smad4, and enter the nucleus to regulate gene expression. Genetic studies for the role of BMPs in osteoblast differentiation have been complicated by at least two factors. First, BMP family members are often expressed in overlapping tissues and may play redundant roles. Second, BMP signaling plays critical roles in cartilage development so that disruption of the pathway in the skeleton often leads to profound early defects that preclude assessment of osteoblast development.54 Nonetheless, by using the Cre-loxP technology a recent study has demonstrated that a critical threshold level of Bmp2/4 signaling was required for trabecular bone formation in the bone marrow cavity, and specifically that the BMP signal controlled the progression from Runx2- to Osterix-positive cells.55 Moreover, although mice lacking only Bmp2 in the limb mesenchyme developed bone during embryogenesis, they exhibited a clear deficit in the mineral density of the long bones shortly after birth, and experienced frequent fractures that failed to heal.56 Contrary to Bmp2 and 4, Bmp3 is a negative regulator of bone mass, as Bmp3null mice contained twice as much trabecular bone volume than the control littermates at 5–6 weeks of age.57 Although the cellular basis for the increased bone mass in Bmp3-null mice remains to be fully elucidated, it is possible that Bmp3 inhibits osteoblastogenesis by counteracting Bmp2, as suggested by in vitro studies.57 Besides its role in osteoblast differentiation, BMP signaling has also been shown to regulate the function of mature osteoblasts. Most notably, genetic deletion of BMPR1A using Og2-Cre (osteocalcin2-Cre)

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The Hedgehog (Hh) family of proteins plays fundamental roles in animal development conserved from flies to humans.42-44 There are three Hh proteins in mammals, namely Indian hedgehog (Ihh), Sonic hedgehog (Shh) and Desert hedgehog (Dhh). Smoothened (Smo), a seven-pass transmembrane protein is indispensable for transducing the Hh signal, but its activity is repressed by another transmembrane protein Pached1 (Ptc1), when Ptc1 is not engaged by Hh proteins. Hh signaling is triggered upon binding of Hh proteins to Ptc1, which relieves the repression of Smo by Ptc1 and activates the intracellular signaling cascade. Activation of the Hh pathway ultimately controls the processing and subcellular localization of the Gli family of transcription factors that in turn modulate expression of downstream target genes. In mammals, three Gli molecules (Gli1-3) collectively mediate Hh signaling. In general, Gli2 and Gli3 function as the predominant activator and repressor, respectively, whereas Gli1, whose expression depends on Hh signaling, plays a secondary role in potentiating Hh response. Indian hedgehog (Ihh), the only family member known to express in the developing skeleton, is indispensable for proper development of the endochondral skeleton. In the developing cartilage, Ihh is primarily expressed by prehypertrophic chondrocytes (chondrocytes immediately prior to hypertrophy) and early hypertrophic chondrocytes; Ihh signals to both immature chondrocytes and the overlying perichondrial cells.45,46 Ihh-null mice exhibited profound defects in both chondrocyte and osteoblast development in the endochondral skeleton.46,47 In particular, loss of Ihh arrested osteoblast development at a primitive stage prior to expression of the earliest known markers including Colα1(I), AP and Runx2, and before the activation of canonical Wnt signaling in the lineage.35 Thus, Ihh functions genetically upstream of canonical Wnt signaling during osteoblast development. By genetically deleting Smo to cell-autonomously remove Hh responsiveness, we found that direct Ihh input was required in the perichondrial osteoprogenitors for development of the osteoblast lineage.47,48 Moreover, through analyses of the Ihh/

BMP Signaling Regulates Osteoblast Differentiation and Function

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Gli3 compound mutant embryos, we show that the control of Ihh over osteoblast differentiation likely requires both derepression of Gli3 repressor and activation of the Gli2 activator.49 The importance of Gli2 activity is consistent with the finding that Gli2-null embryos (lethal at birth) showed impaired osteoblast formation.50 Despite the critical importance of Ihh in embryonic skeletal development, relatively little is known about the role of Hh signaling in the postnatal skeleton. Maeda et al., recently reported that deletion of Ihh from the growth plate chondrocytes in newborn mice resulted in disruption of the growth plate, and continuous loss of trabecular bone in older mice.51 Similarly, pharmacological inhibition of Hh signaling in young mice led to cessation of growth and disruption of bone structure.52 However, these studies did not distinguish direct effects on osteoblasts from those secondary to the chondrocyte defects.

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progression both from Runx2- to Osterix-positive stage,35 and from Osx-positive cells to mature osteoblasts,38 it appears to be dispensable in mature osteoblasts, as its deletion by Col1-Cre or OC-Cre did not affect osteoblasts per se, but instead decreased the expression of Opg by osteoblasts, and therefore indirectly enhanced osteoclast formation.39,40 Whereas a stimulatory role for canonical Wnt signaling in osteoblast differentiation has been supported by genetic studies of β-catenin, Lrp5 and Wnt10b, regulation of Opg expression has not been demonstrated in the studies of either Lrp-5 or Wnt10b. Thus, it remains to be seen whether the Opg regulation by β-catenin in osteoblasts indeed involves the upstream events of Wnt signaling. The positive role of β-catenin signaling in postnatal bone accrual has sparked considerable interest in trying to activate the pathway pharmaceutically to increase bone formation. Additionally, we have recently shown that noncanonical Wnt signaling through G proteincoupled phosphatidylinositol and PKCδ activation also promotes osteoblastogenesis by stimulating the progression from Runx2- to Osterix-positive cells.41 Because this bone-enhancing activity can be uncoupled from canonical Wnt signaling, harnessing this activity may provide bone anabolic benefits while avoiding the potential oncogenic effect of hyperactive canonical Wnt signaling.

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decreased osteoblast function without an obvious effect on osteoblast numbers.58 Similarly, overexpression of noggin, a secreted inhibitor for BMPs, under the osteocalcin promoter, also caused a reduction in osteoblast function and a lower bone mass in postnatal mice.59 Interestingly, the Og2-Cre-mediated BMPR1A-deficient mice also exhibited an decrease in bone resorption when they aged.58 However, it is not known from this study whether this reflected a decrease in osteoclastogenesis in these mice. If so, the underlying mechanism is not clear, because Mishina et al., reported that Rankl expression was not changed in the BMPR1A-deficient bones, whereas others have shown that BMP signaling in osteoblasts induces Opg expression and therefore is expected to inhibit osteoclastogenesis.60 Regardless, it is worth noting that manipulation of BMP signaling in the osteoblast lineage may affect both osteoblasts and osteoclasts.

As discussed above, mouse genetic studies so far have provided compelling evidence that several intercellular signaling pathways control development of the osteoblast lineage. However, most of the studies have relied on tools that disrupt gene activities during embryogenesis, and therefore do not distinguish the embryonic versus postnatal cause of the bone phenotype. This limitation is particularly relevant for studies of bone homeostasis, because early perturbations to the bone marrow are expected to alter the niche environment for mesenchymal progenitors and lead to lasting effects on their fate choice. Future studies that manipulate gene activities specifically in the adult bone are necessary to provide direct answers to the roles of these signaling pathways in adult bone homeostasis. Finally, it remains a greater challenge to understand how the different signals, sometime with opposing properties, integrate to instruct proper fate choice of the progenitors to ensure bone homeostasis in a healthy individual. A recurring theme in bone biology is the exquisite coupling of osteoblast and osteoclast activities. This is best evidenced by the studies of Notch22,24 and Wnt signaling39 in the osteoblast lineage. Moreover, like Notch,23 the other aforementioned signals may also have direct effects on osteoclasts that remain to be elucidated. Therefore, from the perspective of drug development, it is crucial to evaluate the effect of potential anabolic agents on osteoclast formation and function. Strategies to minimize potential undesired increase in bone resorption may be necessary, and could include pulsatile treatment of anabolic agents and combinatorial therapy with anti-resorptive medicine. Finally, targeted delivery of future anabolic agents to the bone may be critical to avoid unintended effects on other tissues. In this regard, the high mineral content and unique surface properties of the bone may offer a great advantage. Overall, understanding the intercellular signals that govern osteoblast differentiation from mesenchymal progenitors may provide potential therapeutic targets for enhancing bone regeneration in osteoporotic patients.

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Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse biological functions.61 The human and mouse genomes contain 22 Fgf and 4 Fgfr genes. Most Fgfs function by binding to and activating cell surface tyrosine kinase Fgf receptors (Fgfrs).62 Fgfs also bind to heparin or heparan sulfate proteoglycans that facilitate the Fgf-binding and activation of Fgfrs. Signaling via Fgfrs is propagated through recruitment and phosphorylation of a variety of signaling proteins, that is triggered by the auto-phosphorylation of the activated receptors and tyrosine-phosphorylation of the closely linked docking proteins.63 As a result, multiple signaling modules including MAPK, PI3K, STAT1 and PKC are activated. Mouse genetic studies have revealed important roles for Fgf signaling in the osteoblast lineage. Mice lacking Fgfr2 selectively in the limb mesenchyme exhibited a decrease in bone mineral density postnatally.64,65 These mice showed a decrease in osteoblast proliferation and a deficit in the mineralizing activity, while maintaining a relatively normal osteoblast differentiation program. On the other hand, mice lacking the mesenchymal splice form of Fgfr2 exhibited a defect in osteoblast differentiation due to a partial loss of Runx2.64 Conversely, mice harboring a gain-of-function mutation in Fgfr2 showed an increase in osteoblast numbers associated with an increase in Runx2 expression and osteoblast differention.66 Likewise, a gainof-function mutation of Fgfr1 stimulated Runx2 expression and enhance osteoblast differentiation in the calvaria, although the status of the long bones was not reported.67 Tissue-specific deletion of Fgfr1 revealed that Fgf1r signaling in osteoprogenitors promotes osteoblast differentiation without affecting Runx2 expression, whereas Fgf1r signaling in mature osteoblasts inhibits the cells’ mineraliszation activity.68 In contrast, mice lacking Fgfr3 showed a decrease in bone mineral density due to defects in mineralization, even though the number of osteoblast was increased.69 Finally, mice lacking Fgf2 showed a marked reduction in total bone mass at the adult stage, due to defects in both osteoblast differentiation and mineralization,70 whereas Fgf18-null embryos exhibited defects in the formation of mature osteoblasts despite normal Runx2 expression.71,72 Thus, Fgf signaling likely promotes osteoblast differentiation, as well as proliferation and mineralization of mature osteoblasts.

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FGF Signaling Regulates Osteoblast Differentiation, Proliferation and Function

Future Directions and Challenges


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