Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

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Osteoblastic cells regulate the haematopoietic stem cell niche

The adult skeleton regenerates by temporary cellular structures that comprise teams of juxtaposed osteoclasts and osteoblasts and replace periodically old bone with new. A considerable body of evidence accumulated during the last decade has shown that the rate of genesis of these two highly specialized cell types, as well as the prevalence of their apoptosis, is essential for the maintenance of bone homeostasis; and that common metabolic bone disorders such as osteoporosis result largely from a derangement in the birth or death of these cells. The purpose of this article is 3-fold: 1) to review the role and the molecular mechanism of action of regulatory molecules, such as cytokines and hormones, in osteoclast and osteoblast birth and apoptosis; 2) to review the evidence for the contribution of changes in bone cell birth or death to the pathogenesis of the most common forms of osteoporosis; and 3) to highlight the implications of bone cell birth and death for a better understanding of the mechanism of action and efficacy of present and future pharmacotherapeutic agents for osteoporosis.

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Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.

The extracellular matrix (ECM), and especially the connective tissue with its collagen, links tissues of the body together and plays an important role in the force transmission and tissue structure maintenance especially in tendons, ligaments, bone, and muscle. The ECM turnover is influenced by physical activity, and both collagen synthesis and degrading metalloprotease enzymes increase with mechanical loading. Both transcription and posttranslational modifications, as well as local and systemic release of growth factors, are enhanced following exercise. For tendons, metabolic activity, circulatory responses, and collagen turnover are demonstrated to be more pronounced in humans than hitherto thought. Conversely, inactivity markedly decreases collagen turnover in both tendon and muscle. Chronic loading in the form of physical training leads both to increased collagen turnover as well as, dependent on the type of collagen in question, some degree of net collagen synthesis. These changes will modify the mechanical properties and the viscoelastic characteristics of the tissue, decrease its stress, and likely make it more load resistant. Cross-linking in connective tissue involves an intimate, enzymatical interplay between collagen synthesis and ECM proteoglycan components during growth and maturation and influences the collagen-derived functional properties of the tissue. With aging, glycation contributes to additional cross-linking which modifies tissue stiffness. Physiological signaling pathways from mechanical loading to changes in ECM most likely involve feedback signaling that results in rapid alterations in the mechanical properties of the ECM. In developing skeletal muscle, an important interplay between muscle cells and the ECM is present, and some evidence from adult human muscle suggests common signaling pathways to stimulate contractile and ECM components. Unaccostumed overloading responses suggest an important role of ECM in the adaptation of myofibrillar structures in adult muscle. Development of overuse injury in tendons involve morphological and biochemical changes including altered collagen typing and fibril size, hypervascularization zones, accumulation of nociceptive substances, and impaired collagen degradation activity. Counteracting these phenomena requires adjusted loading rather than absence of loading in the form of immobilization. Full understanding of these physiological processes will provide the physiological basis for understanding of tissue overloading and injury seen in both tendons and muscle with repetitive work and leisure time physical activity.

Role of Extracellular Matrix in Adaptation of Tendon and Skeletal Muscle to Mechanical Loading

Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.

Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours

The past 15 years have witnessed tremendous progress in the molecular understanding of osteoblasts, the main bone-forming cells in the vertebrate skeleton In particular, all of the major developmental signals (including WNT and Notch signalling), along with an increasing number of transcription factors (such as RUNX2 and osterix), have been shown to regulate the differentiation and/or function of osteoblasts As evidence indicates that osteoblasts may also regulate the behaviour of other cell types, a clear understanding of the molecular identity and regulation of osteoblasts is important beyond the field of bone biology

Building strong bones: molecular regulation of the osteoblast lineage

Notch receptors are determinants of cell fate decisions. To define the role of Notch in the adult skeleton, we created transgenic mice overexpressing the Notch intracellular domain(NICD)underthecontrolofthetypeIcollagenpromoter. First-generationtransgenicsweresmallandosteopenic.Bone histomorphometry revealed that NICD caused a decrease in bone volume, secondary to a reduction in trabecular number; osteoblast and osteoclast number were decreased. Low fertility of founder mice and lethality of young pups did not allow the complete establishment of transgenic lines. To characterize the effect of Notch overexpression in vitro, NICD was induced in osteoblasts and stromal cells from Rosa notch mice, in which a STOP cassette flanked by lox P sites is upstream of NICD, by transduction with an adenoviral vector expressing Cre recombinase (Cre) under the control of the cytomegalovirus (CMV) promoter (Ad-CMV-Cre). NICD impaired osteoblastogenesis and inhibited Wnt/-catenin signaling. To determine the effects of notch1 deletion in vivo, mice in which notch1 was flanked bylox P sequences (notch1 loxP/loxP ) were mated with mice expressing Cre recombinase under the control of the osteocalcin promoter. Conditional null notch1 mice had no obvious skeletal phenotype, possibly because of rescue by notch2; however, 1-month-old females exhibited a modest increase in osteoclast surface and eroded surface. Osteoblasts from notch1 loxP/loxP mice, transduced with Ad-CMV-Cre and transfected with Notch2 small interfering RNA, displayed increased alkaline phosphatase activity. In conclusion, Notch signaling in osteoblasts causes osteopenia and impairs osteoblastogenesis by inhibiting the Wnt/-catenin pathway. (Endocrinology 149: 3890–3899, 2008)

Notch Inhibits Osteoblast Differentiation And Causes Osteopenia

Glucocorticoids have profound effects on bone formation, decreasing IGF I transcription in osteoblasts, but the mechanisms involved are poorly understood. We previously showed that the bp +34 to+ 192 region of the rat IGF I exon 1 promoter was responsible for repression of IGF I transcription by cortisol in cultures of osteoblasts from fetal rat calvariae (Ob cells). Here, site-directed mutagenesis was used to show that a binding site for members of the CAAT/enhancer binding protein family of transcription factors, within the +132 to +158 region of the promoter, mediates this glucocorticoid effect. EMSAs demonstrated that cortisol increased binding of osteoblast nuclear proteins to the +132 to +158 region of the IGF I promoter. Supershift assays showed that CAAT/enhancer binding protein α, β, and δ interact with this sequence, and binding of CAAT/enhancer binding protein δ, in particular, was increased in the presence of cortisol. Northern blot analysis showed that CAAT/enhancer binding protein δ and β tr...

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Glucocorticoid Suppression of IGF I Transcription in Osteoblasts

http://www.jbc.org/content/282/43/31549.full.pdf

Conditional deletion of gremlin causes a transient increase in bone formation and bone mass

Glucocorticoids have important effects on osteoblastic replication, differentiation, and function, and the Notch family of receptors is considered to play a role in osteoblastic cell differentiation. We postulated that cortisol could regulate Notch and Notch ligand expression in osteoblastic cells, providing an additional mechanism by which glucocorticoids could regulate osteoblastic differentiation. We examined the expression and regulation of Notch1, 2, 3, and 4 and their ligands Jagged 1 and 2 and Delta 1 and 3 by cortisol in cultures of osteoblastic MC3T3-E1 cells. Cortisol caused a time-dependent increase in Notch1 and 2 mRNA levels in MC3T3 cells. Notch3 and 4 were not detected in the presence or absence of cortisol. MC3T3 cells expressed Delta 1 and Jagged 1 but not Jagged 2 or Delta 3 mRNAs, and cortisol did not have a substantial effect on the expression of any of these ligands. Cortisol increased the rate of Notch1 and 2 transcription and, in transcriptionally arrested cells, did not modify the decay of the transcripts, indicating a transcriptional level of control. In conclusion, cortisol stimulates Notch1 and 2 transcription in osteoblasts. Since Notch signaling appears to play a negative role in osteoblastic differentiation, its increased expression could be relevant to the actions of cortisol in bone.

Cortisol regulates the expression of Notch in osteoblasts.

Platelet-derived Growth Factor Induces Interleukin-6 Transcription in Osteoblasts through the Activator Protein-1 Complex and Activating Transcription Factor-2

Deena Durant

Papers

Notch Inhibits Osteoblast Differentiation And Causes Osteopenia

Glucocorticoid Suppression of IGF I Transcription in Osteoblasts

Conditional deletion of gremlin causes a transient increase in bone formation and bone mass

Cortisol regulates the expression of Notch in osteoblasts.

Platelet-derived Growth Factor Induces Interleukin-6 Transcription in Osteoblasts through the Activator Protein-1 Complex and Activating Transcription Factor-2