Osteogenic growth peptide: from concept to drug design

Recently, the osteogenic growth peptide (OGP) and its C-terminal pentapeptide H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)] have attracted considerable clinical interest as bone anabolic agents and hematopoietic stimulators. They are present in mammalian serum in micromolar concentrations, increase bone formation and trabecular bone density, and stimulate fracture healing when administered to mice and rats. In cultures of osteoblastic and other bone marrow stromal cells, derived from human and other mammalian species, OGP regulates proliferation, alkaline phosphatase activity and matrix mineralization via an autocrine/paracrine mechanism. In vivo it also regulates the expression of type I collagen and the receptor for basic fibroblast growth factor. In addition, OGP and OGP(10-14) enhance hematopoiesis, including the stimulation of bone marrow transplant engraftment and hematopoietic regeneration after ablative chemotherapy. Apparently, the hematopoietic effects of these peptides are secondary to their effect on the bone marrow stroma. Detailed structure-activity relationship study identified the side chains of Tyr(10) and Phe(12) as the principal pharmacophores for OGP-like activity. Recently, it has been demonstrated that several cyclostereoisomers of OGP(10-14), including the analogue retro-inverso (Gly-Gly-D-Phe-Gly-D-Tyr), share the full spectrum of OGP-like bioactivities. Taken together, OGP represents an interesting case of a "housekeeping" peptide that plays an important role in osteogenesis and hematopoiesis, and interacts with its putative macromolecular target via distinct pharmacophores presented in a specific spatial organization.     

Mitogenic G(i) protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: activation by C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)] and attenuation of activation by cAMP

In osteogenic and other cells the mitogen-activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP-binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C-terminal pentapeptide OGP(10-14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10-14), but not OGP, enhance MAP kinase activity in a time-dependent manner. The OGP(10-14)-induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a G(i) protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide-activated, mitogenic Gi protein-MAP kinase-signaling cascade. Forskolin and dBu(2)-cAMP abrogated the OGP(10-14)-stimulated proliferation, but induced only 50% inhibition of the OGP(10-14)-mediated MAP kinase activation, suggesting additional MAP kinase-dependent, OGP(10-14)-regulated, cellular functions. Finally, it is demonstrated that OGP(10-14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP-OGPBP complexes.     

The construction of high efficiency human bone marrow tissue ex vivo

The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Osteogenic growth peptide C-terminal pentapeptide [OGP(10-14)] acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

Cumulative evidence indicates that bone marrow mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating to osteogenic and adipogenic lineages when stimulated under appropriate conditions. Whether OGP(10-14) directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. In the present study, we investigated the roles of OGP(10-14) in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that OGP(10-14) promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. OGP(10-14) increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of core-binding factor 1 (cbfa1). In contrast, OGP(10-14) decreased adipocyte numbers and inhibited adipocyte-specific mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2). These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is regulated by OGP(10-14).     

Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal

The Mixed Lineage Leukemia (Mll) gene is a homolog of Drosophila Trithorax commonly rearranged in infant leukemia. Comprehensive analysis of the role of Mll in hematopoiesis in fetal and adult knockout mice has been prevented by the lethality of Mll(-/-) mice. We have established a conditional deletion model that allows us to study adult hematopoiesis in the absence of Mll. In this study, Mll(-/-) embryos survive to E16.5 and have reduced numbers of HSCs. The quiescent fraction of these HSCs is greatly reduced, and they are unable to compete with wild-type cells in transplantation assays. Mice with Mll expression conditionally deleted in the hematopoietic system have grossly normal hematopoiesis in bone marrow, thymus, and spleen. However, transplanted Mll-deficient bone marrow cells are highly compromised in their ability to competitively reconstitute irradiated recipients. These results suggest a critical role for Mll in regulating stem cell self-renewal.     

Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCK pathway

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vitro studies have demonstrated that OGP directly regulates the bone marrow mesenchymal stem cells' (BMSCs) differentiation into osteoblasts. However, the exact mechanism of this process remains unknown. In the present study, we investigated the role of RhoA/ROCK signaling in differentiation along this lineage using human BMSCs. OGP treatment increased the mRNA level of bone morphogenetic protein-2 and alkaline phosphatase activity after osteogenic induction. Analysis of BMSCs induced in the presence of OGP revealed an increase in RhoA activity, and phosphorylation of FAK and cofilin. The ROCK-specific inhibitors, Y27632, blocked the OGP-induced regulation of BMSC differentiation. Taken together, these data suggest that OGP not only acts on BMSCs to stimulate osteogenic differentiation, but also in a dose-dependent manner, and this effect is mediated via the activation of RhoA/ROCK pathway.     

Osteogenic growth peptide effects on primary human osteoblast cultures: potential relevance for the treatment of glucocorticoid-induced osteoporosis

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vivo studies on animals have demonstrated that the synthetic peptide OGP (10-14), reproducing the OGP C-terminal active portion [H-Tyr-Gly-Phe-Gly-Gly-OH] increases bone formation, trabecular bone density and fracture healing. In vitro studies performed on cellular systems based on osteoblastic-like cell lines or mouse stromal cells, have demonstrated that OGP (10-14) increases osteoblast proliferation, alkaline phosphatase (ALKP) activity and matrix synthesis and mineralization. In view of a potential application of OGP (10-14) in clinical therapy, we have tested different concentrations of OGP (10-14) on primary human osteoblast (hOB) cultures. We have observed significant increases of hOB proliferation (+35%), ALKP activity (+60%), osteocalcin secretion (+50%), and mineralized nodules formation (+49%). Our experimental model based on mature hOBs was used to investigate if OGP (10-14) could prevent the effects on bone loss induced by sustained glucocorticoid (GC) treatments. A strong decrease in bone formation has been attributed to the effects of GCs on osteoblastogenesis and osteocyte apoptosis, while an increase in bone resorption was due to a transient osteoblastic stimulation, mediated by the OPG/RANKL/RANK system, of osteoclasts recruitment and activation. Moreover, GCs act on hOBs decreasing the release of osteoprotegerin (OPG) a regulator of the RANKL/RANK interaction. Here, we provide evidences that OGP (10-14) inhibits hOB apoptosis induced by an excess of dexamethasone (-48% of apoptotic cells). Furthermore, we show that OGP (10-14) can increase OPG secretion (+20%) and can restore the altered expression of OPG induced by GCs to physiological levels. Our results support the employment of OGP (10-14) in clinical trials addressed to the treatment of different bone remodeling alterations including the GC-induced osteoporosis.     

The cholinergic system is involved in regulation of the development of the hematopoietic system

Gene expression profiling demonstrated that components of the cholinergic system, including choline acetyltransferase, acetylcholinesterase and nicotinic acetylcholine receptors (nAChRs), are expressed in embryonic stem cells and differentiating embryoid bodies (EBs). Triggering of nAChRs expressed in EBs by nicotine resulted in activation of MAPK and shifts of spontaneous differentiation toward hemangioblast. In vivo, non-neural nAChRs are detected early during development in fetal sites of hematopoiesis. Similarly, in vivo exposure of the developing embryo to nicotine resulted in higher numbers of hematopoietic progenitors in fetal liver. However postpartum, the number of hematopoietic stem/progenitor cells (HSPC) was decreased, suggesting an impaired colonization of the fetal bone marrow with HSPCs. This correlated with increased number of circulating HSPC and decreased expression of CXCR4 that mediates migration of circulating cells into the bone marrow regulatory niche. In addition, protein microarrays demonstrated that nicotine changed the profile of cytokines produced in the niche. While the levels of IL1alpha, IL1beta, IL2, IL9 and IL10 were not changed, the production of hematopoiesis-supportive cytokines including G-CSF, GM-CSF, IL3, IL6 and IGFBP-3 was decreased. This correlated with the decreased repopulating ability of HSPC in vivo and diminished hematopoietic activity in bone marrow cultures treated with nicotine. Interestingly, nicotine stimulated the production of IL4 and IL5, implying a possible role of the cholinergic system in pathogenesis of allergic diseases. Our data provide evidence that the nicotine-induced imbalance of the cholinergic system during gestation interferes with normal development and provides the basis for negative health outcomes postpartum in active and passive smokers.     

In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.     

SDF-1 and CXCR4 in normal and malignant hematopoiesis

Over recent years it has become apparent that the chemokine SDF-1 and its receptor CXCR4 play pivotal roles in normal hematopoiesis. They are essential for the normal ontogeny of hematopoiesis during embryogenesis and continue to play a key role in retaining hematopoietic progenitors within the bone marrow microenvironment in the adult. As a result of this role disruption of SDF-1/CXCR4 interactions results in mobilization of hematopoietic progenitors and standard mobilization protocols disrupt this axis. Similarly SDF-1/CXCR4 interactions are required for homing and engraftment of hematopoietic stem cells during transplantation. SDF-1 regulates the localisation of leukemic cells and like their normal counterparts most leukemic cells respond to SDF-1 with increased adhesion, survival and proliferation. However in some instances leukemic cell responses to SDF-1 can be disregulated, the impact of which on the progression of disease in not known. In this review we discuss the pleiotropic roles of SDF-1/CXCR4 interactions in human hematopoietic stem cell ontogeny, bone marrow homing and engraftment, mobilization and how these interactions impact on malignant hematopoiesis.     

Oncostatin M regulates mesenchymal cell differentiation and enhances hematopoietic supportive activity of bone marrow stromal cell lines

Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.     

Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.     

Akt signaling regulates side population cell phenotype via Bcrp1 translocation

Akt is an important regulator of cell survival, growth, and glucose metabolism in many cell types, but the role of this signaling molecule in hematopoietic stem cells is poorly defined. Side population (SP) cells are enriched for hematopoietic stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342. Bone marrow from Akt1-null mice exhibited a reduced SP fraction. However, bone marrow cellularity, growth factor-responsive progenitor cultures, and engraftable stem cells were normal in these mice. Treatment of bone marrow with LY294002, an inhibitor of the Akt effector protein phosphatidylinositol 3-kinase, led to a reversible loss of the SP fraction. Bcrp1, which encodes the Hoechst dye transporter, was translocated from the membrane to the intracellular compartment under conditions that promote the SP-depleted state. Lentivirus-mediated overexpression of Akt1 in bone marrow markedly increased the SP fraction, whereas there was no effect on bone marrow from Bcrp(-/-) mice. These data suggest that Akt signaling modulates the SP cell phenotype by regulating the expression of Bcrp1.     

Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells.

The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10-14). The OGP(10-14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10-14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP-OGP binding protein (OGPBP) complex formation, sOGP(10-14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10-14) is a naturally occurring human and murine mitogen. In addition, the data suggests that the OGP(10-14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP-OGPBP complexes.     

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.     

Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.