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.     

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).     

Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells

Transforming growth factor beta (TGF-beta) activates Ras/MAPK signaling in many cell types. Because TGF-beta and BMP-2 exert similar effects, we examined if this signaling is stimulated by both factors and analyzed the relationship between this signaling and the Smads in osteoblasts. BMP-2 and TGF-beta stimulated Ras, MAPK, and AP-1 activities. The DNA binding activities of c-Fos, FosB/Delta FosB, Fra-1, Fra-2, and JunB were up-regulated whereas JunD activity was decreased. c-Fos, FosB/Delta FosB, and JunB were associated with Smad4. The stimulation of AP-1 by BMP-2 and TGF-beta was dependent on Smad signaling, and anti-Smad4 antibody interfered with AP-1 activity. Thus, BMP-2 and TGF-beta activate both Ras/MAPK/AP-1 and Smad signaling in osteoblasts with Smads modulating AP-1 activity. To determine the roles of MAPK in BMP-2 and TGF-beta function, we analyzed the effect of ERK and p38 inhibitors on the regulation of bone matrix protein expression and JunB and JunD levels by these two factors. ERK and p38 mediated TGF-beta suppression of osteocalcin and JunD as well as stimulation of JunB. p38 was essential in BMP-2 up-regulation of type I collagen, fibronectin, osteopontin, osteocalcin, and alkaline phosphatase activity whereas ERK mediated BMP-2 stimulation of fibronectin and osteopontin. Thus, ERK and p38 differentially mediate TGF-beta and BMP-2 function in osteoblasts.     

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.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Direct production of gene-targeted mice from ES cells by nuclear transfer and gene transmission to their progeny.

In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP-/+ mice were composed of 9 OGP-/-, 18 OGP-/+, and 10 OGP+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP-/-, 12 OGP-/+, and 4 OGP+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.     

Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.     

Osteogenic growth peptide enhances the proliferation of bone marrow mesenchymal stem cells from osteoprotegerin-deficient mice by CDK2/cyclin A

To promote bone formation is one of the fundamental strategies in osteoporosis treatment and fractures repair. As one of the stimulators on bone formation, osteogenic growth peptide (OGP) increases both proliferation and differentiation of the osteoblasts in vitro and in vivo, in which osteoprotegerin (OPG) has been suggested being involved. In this study, we evaluated the effects of OGP on bone marrow mesenchymal stem cells (MSCs) from OPG-deficient mice in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase (ALP) activity assay, real-time polymerase chain reaction, and western blot analysis. Results showed that OGP stimulated MSC proliferation and increased the expression of CDK2 and cyclin A in MSCs both at mRNA and protein levels. However, no differentiative effect of OGP was shown as ALP activity and the expression levels of Runx2 and Osterix were not increased significantly by OGP. Our study suggested that OGP may increase the bone formation in OPG-deficient mice by stimulating MSC proliferation rather than differentiation, and probably by triggering CDK2/cyclin A pathway.     

Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)].

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10-14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or D-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with L- or D-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and L- or D-Ala substitution of Gly13 resulted in 70-97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10-14).     

MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells

Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen- and stress-activated protein kinase (MSK)1-/- and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser-133 and ATF1 at Ser-63 in wild type cells and this was abolished by inhibition of the mitogen-activated protein kinase cascade. In contrast, the TPA- and EGF-induced phosphorylation of CREB/ATF1 was barely detectable in MSK1-/- cells. However, basal and forskolin-induced phosphorylation was similar, indicating that the MSK1 'knockout' did not prevent CREB phosphorylation by cyclic AMP-dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells.