Paul G. Genever

Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells.

Through their broad differentiation potential, mesenchymal stem cells (MSCs) are candidates for a range of therapeutic applications, but the precise signaling pathways that determine their differentiated fate are not fully understood. Evidence is emerging that developmental signaling cues may be important in regulating stem cell self‐renewal and differentiation programs. Here we have identified a consistent expression profile of Wnt signaling molecules in MSCs and provide evidence that an endogenous canonical Wnt pathway functions in these cells.

Wnt signalling in osteoblasts regulates expression of the receptor activator of NFκB ligand and inhibits osteoclastogenesis in vitro

Reports implicating Wnt signalling in the regulation of bone mass have prompted widespread interest in the use of Wnt mimetics for the treatment of skeletal disorders. To date much of this work has focused on their anabolic effects acting on cells of the osteoblast lineage. In this study we provide evidence that Wnts also regulate osteoclast formation and bone resorption, through a mechanism involving transcriptional repression of the gene encoding the osteoclastogenic cytokine receptor activator of NFκB ligand (RANKL or TNFSF11) expressed by osteoblasts. In co-cultures of mouse mononuclear spleen cells and osteoblasts, inhibition of GSK3β with LiCl or exposure to Wnt3a inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells compared with controls. However, these treatments had no consistent effect on the differentiation, survival or activity of osteoclasts generated in the absence of supporting stromal cells. Activation of Wnt signalling downregulated RANKL mRNA and protein expression, and overexpression of fulllength β-catenin, but not transcriptionally inactive β-catenin ΔC(695-781), inhibited RANKL promoter activity. Since previous studies have demonstrated an absence of resorptive phenotype in mice lacking LRP5, we determined expression of a second Wnt co-receptor LRP6 in human osteoblasts, CD14+ osteoclast progenitors and mature osteoclasts. LRP5 expression was undetectable in CD14-enriched cells and mature human osteoclasts, although LRP6 was expressed at high levels by these cells. Our evidence of Wnt-dependent regulation of osteoclastogenesis adds to the growing complexity of Wnt signalling mechanisms that are now known to influence skeletal function and highlights the requirement to develop novel therapeutics that differentially target anabolic and catabolic Wnt effects in bone.

Mechanically regulated expression of a neural glutamate transporter in bone: A role for excitatory amino acids as osteotropic agents?

Without habitual exercise, bone is lost from the skeleton. Interactions between the effects of loading of bone and other osteotropic influences are thought to regulate bone mass. In an attempt to identify potential targets for therapeutic manipulation of bone mass, we used differential RNA display to investigate early changes in osteocyte gene expression following mechanical loading of rat bone in vivo. One gene found to be down-regulated by loading had high homology to a glutamate/ aspartate transporter (GLAST) previously identified only the mammalian CNS. RT-PCR analysis using primers targeted to the coding region of the published GLAST sequence amplified identical products from bone and brain (but not a range of other tissues). The amplicons were sequenced and found to be identical to the published CNS GLAST sequence. Northern analysis confirmed expression of GLAST mRNA in bone and brain, but not other tissues. In situ hybridization localized GLAST mRNA expression in rat bone to osteoblasts and osteocytes. A GLAST antibody localized high levels of protein expression to osteoblasts, and newly incorporated osteocytes. Interestingly, older osteocytes also expressed detectable levels of GLAST. Another neural glutamate transporter, GLT-1 was immunolocalized to the pericellular region of mononuclear bone marrow cells, while a further antibody to the EAAC-1 transporter failed to bind to bone cells. Five days after loading, GLAST protein expression was undetectable in osteocytes of loaded bone but present in control nonloaded sections, confirming the downregulation detected by differential display. On quiescent periosteal surfaces, GLAST expression was almost absent, while on surfaces where loading had induced cellular proliferation and bone formation, GLAST protein expression was elevated. In the CNS, the expression of glutamate transporters on neuronal membranes is associated with reuptake of released neurotransmitters at synapses, where they have a role in the termination of transmitter action. In this study, we describe for the first time, the expression of GLAST (and GLT-1) in bone, raising the possibility that excitatory amino acids may have a role in paracrine intercellular communication in bone. Manipulation of bone cell function by moderators of glutamate action could therefore provide novel treatments for bone diseases such as osteoporosis.

Expression of an N-Methyl-D-Aspartate-Type Receptor by Human and Rat Osteoblasts and Osteoclasts Suggests a Novel Glutamate Signaling Pathway in Bone

Signaling between the various types of cells found in bone is responsible for controlling the activity of osteoblasts and osteoclasts, and therefore the regulation of bone mass. Our identification of a neuronal glutamate transporter in osteoblasts and osteocytes suggests the possibility that bone cells may use the excitatory amino acid glutamate as a signaling molecule. In these studies we report the expression of different subtypes of glutamate receptors in osteoblasts and osteoclasts in vitro and in vivo. We have identified expression in human and rat bone cells of N-methyl-D-aspartate receptor-1 (NMDAR-1) and 2D subunits and PSD-95, the NMDA receptor clustering protein associated with signaling in the central nervous system. In situ hybridization and immunohistochemistry localized NMDAR-1 expression to osteoblasts and osteoclasts in human tissue sections. These findings strengthen the suggestion that glutamate is involved in signaling between bone cells.

A role for N-cadherin in the development of the differentiated osteoblastic phenotype.

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast‐derived N‐cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N‐cadherin expression was localized prominently in well‐differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS‐2 and rat ROS osteoblast‐like cells showed a relationship between N‐cadherin messenger RNA expression and cell‐to‐cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His‐Ala‐Val (HAV) adhesion motif of N‐cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell‐to‐cell contact in rat osteoblastic TRAB‐11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS‐2 cells. Transient transfection of SaOS‐2 cells with a dominant‐negative N‐cadherin mutant (NCADΔC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast‐derived N‐cadherin was inhibited by either treatment with HAV or transfection with NCADΔC. Together, these results strongly support a role for N‐cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation. (J Bone Miner Res 2000;15:198–208)

Evidence for Targeted Vesicular Glutamate Exocytosis in Osteoblasts

Regulated intercellular signaling is essential for the maintenance of bone mass. In recent work we described how osteoblasts and osteoclasts express functional receptors for the excitatory amino acid, glutamate, indicating that a signaling pathway analogous to synaptic neurotransmission exists in bone. Here, we show that osteoblasts also express the essential molecular framework for regulated glutamate exocytosis to occur as is present in presynaptic neurons. A combination of reverse transcription-polymerase chain reaction (RT-PCR) and northern and western blotting is used to show expression of the target membrane-SNARE (soluble NSF attachment protein receptor), proteins SNAP-25 and syntaxin 4 and the vesicular-SNARE protein VAMP (synaptobrevin), the minimum molecular requirements for core exocytotic complex formation. Immunofluorescent localizations reveal peripheral SNAP-25 expression on osteoblastic cells, particularly at intercellular contact sites, colocalizing with immunoreactive glutamate and the synaptic vesicle-specific protein, synapsin I. We also identify multiple accessory proteins associated with vesicle trafficking, including munc18, rSec8, DOC2, syntaxin 6, and synaptophysin, which have varied roles in regulated glutamate exocytosis. mRNA for the putative Ca(2+)-dependent regulators of vesicle recycling activity, synaptotagmin I (specialized for fast Ca(2+)-dependent exocytosis as seen in synaptic neurotransmission), and the GTP-binding protein Rab3A are also identified by northern blot analysis. Finally, we demonstrate that osteoblastic cells actively release glutamate in a differentiation-dependent manner. These data provide compelling evidence that osteoblasts are able to direct glutamate release by regulated vesicular exocytosis, mimicking presynaptic glutamatergic neurons, showing that a process with striking similarity to synaptic neurotransmission occurs in bone.

Regulation of spontaneous glutamate release activity in osteoblastic cells and its role in differentiation and survival: evidence for intrinsic glutamatergic signaling in bone.

Maintenance of bone mass depends on numerous osteoblast‐derived autocrine and paracrine signaling factors that ensure the coupled resorptive and formative activity of osteoclasts and osteoblasts. Here, we provide the first evidence that osteoblasts actively secrete glutamate, an amino acid neurotransmitter found at excitatory synapses in the central nervous system, complementing previous reports of functional glutamate receptor expression by bone cells. Several osteoblastic cell‐types spontaneously released between 2–7 nmoles glutamate per mg protein, equivalent to or greater than reported levels of glutamate release from depolarized neurons. Osteoblastic glutamate exocytosis appeared dependent on an AMPA‐type glutamate autoreceptor, and addition of depolarizing concentrations of KCl caused significant calcium‐dependent inhibition of glutamate release. Levels of exocytosed and intracellular free glutamate, and susceptibility to depolarization‐induced inhibition of glutamate release, increased during osteoblastic differentiation of MC3T3‐E1 cells. Pharmacological inhibition of glutamate release with riluzole (1–25 µM) significantly inhibited differentiation and induced morphological and biochemical characteristics of apoptosis in osteoblastic cells. Exogenous glutamate application increased survival rates of osteoblasts grown in serum‐free medium, and proinflammatory cytokines (tumor necrosis factor–α and interferon–γ) significantly inhibited osteoblastic glutamate release. These findings provide evidence for an intrinsic synaptic‐like glutamatergic signaling network in bone that is essential for in vitro osteoblast differentiation and survival.

Advancement of mesenchymal stem cell therapy in solid organ transplantation (MISOT).

There is evolving interest in the use of mesenchymal stem cells (MSC) in solid organ transplantation. Pre-clinical transplantation models show efficacy of MSC in prolonging graft survival and a number of clinical studies are planned or underway. At a recent meeting of the MISOT consortium (MSC In Solid Organ Transplantation) the advances of these studies were evaluated and mechanisms underlying the potential effects of MSC discussed. Continued discussion is required for definition of safety and eventually efficacy endpoints for MSC therapy in solid organ transplantation.

Localization of ADAM10 and notch receptors in bone

In Drosophila melanogaster, the role of the metallodisintegrin, Kuzbanian (kuz), is thought to involve activation of the Drosophila Notch receptor that plays a role in cell-fate determination during neurogenesis and myoblast differentiation. To understand the possible function(s) of a-disintegrin and metalloproteinase (ADAM10), the mammalian ortholog of kuz, in the skeleton, we studied its expression as well as the messenger RNA (mRNA) encoding one candidate substrate, the mammalian Notch2 receptor in bone, bone cells, and cartilage. In sections of neonatal rat tibiae, ADAM10 is expressed in specific regions of articular cartilage and metaphyseal bone. Expression of ADAM10 in articular cartilage occurs predominantly in superficial chondrocytes and becomes more sporadic with increasing distance from the articular surface. In bone, ADAM10 is expressed by periosteal cells, osteoblasts, and osteocytes at locations of active bone formation. Osteoclasts did not express ADAM10. Notch2 mRNA expression was not detectable in superficial chondrocytes. However it colocalized at all sites of ADAM10 expression in bone cells. In vitro, both primary human osteoblasts and osteoblast cell lines expressed a single 4.5 kb and 7.5 kb transcript of ADAM10 and the Notch2 receptor homolog, respectively. Subcellular localization of the ADAM10 protein in MG-63 cells was determined using immunofluorescent techniques. These observations showed clearly that the ADAM10 protein was expressed in the trans-Golgi network and on the plasma membrane. Western blot analysis of fractionated cells showed that, in the plasma membrane fraction, the previously characterized 58 kDa and 56 kDa isoforms were present, whereas, in the trans-Golgi network, the ADAM10 protein was present in several additional bands, possibly indicative of further interdomain processing of the ADAM10 protein. The metallodisintegrins (ADAMs) have several putative functions, including modulation of cell adhesion, membrane-associated proteolysis, and cell-cell signaling. These observations suggest that, in bone but not cartilage, ADAM10 has catalytic activity within the transGolgi network and may play a role in the activation of Notch receptor homologs. This implicates ADAM10 in cell-fate determination of osteoblast progenitor cells, possibly during skeletal development and normal bone remodeling. Plasma-membrane-associated ADAM10 may confer alternative functions.

SOX9 determines RUNX2 transactivity by directing intracellular degradation.

Mesenchymal stem cell differentiation is controlled by the cooperative activity of a network of signaling mechanisms. Among these, RUNX2 and SOX9 are the major transcription factors for osteogenesis and chondrogenesis, respectively. Their expression is overlapped both temporally and spatially during embryogenesis. Here we have demonstrated that RUNX2 and SOX9 physically interact in intact cells and have confirmed that SOX9 can inhibit the transactivation of RUNX2. In addition, RUNX2 exerts reciprocal inhibition on SOX9 transactivity. In analyses of the mechanism by which SOX9 regulated RUNX2 function, we demonstrated that SOX9 induced a dose‐dependent degradation of RUNX2. Although RUNX2 is normally degraded by the ubiquitin‐proteasome pathway, we found that SOX9‐mediated degradation was proteasome‐independent but phosphorylation‐dependent and required the presence of the RUNX2 C‐terminal domain, which contains a nuclear matrix targeting sequence (NMTS). Furthermore, SOX9 was able to decrease the level of ubiquitinated RUNX2 and direct RUNX2 to the lysosome for degradation. SOX9 also preferentially directed β‐catenin, an intracellular mediator of canonical Wnt signaling, for lysosomal breakdown. Consequently, the mechanisms by which SOX9 regulates RUNX2 function may underlie broader signaling pathways that can influence osteochondrogenesis and mesenchymal fate.