E1 Cells Research Articles

Palladin, an actin-associated protein, is required for adherens junction formation and intercellular adhesion in HCT116 colorectal cancer cells

Palladin is a scaffold protein involved in the formation of actin-associated protein complexes. Gene expression array analysis on the poorly metastatic HCT116 colon cancer cell line and a metastatic derivative cell line (E1) with EMT (epithelial-mesenchymal transition) features showed a down-regulation of palladin gene expression in the latter. Knockdown of palladin expression in the HCT116 cells suppressed junctional localization of E-cadherin, reduced intercellular adhesion and collective cell migration, showing that palladin plays an important role in maintaining the integrity of adherens junctions. The acquisition of the EMT features by the E1 cell line was dependent on the Erk pathway. Inhibition of this pathway by U0126 treatment in E1 cells resulted in the re-expression of palladin, relocalization of E-cadherin to the adherens junctions and a reversal of EMT features. The re-establishment of intercellular adhesion was dependent on palladin expression. The down-regulation of palladin was also observed in poorly-differentiated tumor tubules and dissociated tumor cells that have undergone de-differentiation in human primary colon tumors. Our data show that palladin is an integral component of adherens junctions and plays a role in the localization of E-cadherin to the junctions. The loss of palladin may be an integral part of EMT, an early step in the metastatic spread of colon carcinoma.

An efficient surface modification using 2-methacryloyloxyethyl phosphorylcholine to control cell attachment via photochemical reaction in a microchannel

This report describes a direct approach for cell micropatterning in a closed glass microchannel. To control the cell adhesiveness inside the microchannel, the application of an external stimulus such as ultraviolet (UV) was indispensible. This technique focused on the use of a modified 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to be a non-biofouling compound that is a photocleavable linker (PL), to localize cells via connection to an amino-terminated silanized surface. Using UV light illumination, the MPC polymer was selectively eliminated by photochemical reaction that controlled the cell attachment inside the microchannel. For suitable cell micropatterning in a microchannel, the optimal UV illumination time and concentration for cell suspension were investigated. After selective removal of the MPC polymer through the photomask, MC-3T3 E1 cells and vascular endothelial cells (ECs) were localized only to the UV-exposed area. In addition, the stability of patterned ECs was also confirmed by culturing for 2 weeks in a microchannel under flow conditions. Furthermore, we employed two different types of cells inside the same microchannel through multiple removal of the MPC polymer. ECs and Piccells were localized in both the upper and down streams of the microchannel, respectively. When the ECs were stimulated by adenosine triphosphate (ATP), NO was secreted from the ECs and could be detected by fluorescence resonance energy transfer (FRET) in Piccells, which is a cell-based NO indicator. This technique can be a powerful tool for analyzing cell interaction research.

A simple PDMS-based microfluidic channel design that removes bubbles for long-term on-chip culture of mammalian cells

This report shows methods to fabricate polydimethylsiloxane (PDMS) microfluidic systems for long-term (up to 10 day) cell culture. Undesired bubble accumulation in microfluidic channels abruptly changes the microenvironment of adherent cells and leads to the damage and death of cells. Existing bubble trapping approaches have drawbacks such as the need to pause fluid flow, requirement for external vacuum or pressure source, and possible cytotoxicity. This study reports two kinds of integrated bubble trap (IBT) which have excellent properties, including simplicity in structure, ease in fabrication, no interference with the flow, and long-term stability. IBT-A provides the simplest solution to prevent bubbles from entering microfluidic channels. In situ time-lapse imaging experiments indicate that IBT-B is an excellent device both for bubble trapping and debubbling in cell-loaded microfluidics. MC 3T3 E1 cells, cultured in a long and curved microfluidic channel equipped with IBT-B, showed high viability and active proliferation after 10 days of continuous fluid flow. The comprehensive measures taken in our experiments have led to successful long-term, bubble-free, on-chip culture of cells.

Fhit Interaction with Ferredoxin Reductase Triggers Generation of Reactive Oxygen Species and Apoptosis of Cancer Cells

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

Microarray analysis of thapsigargin — induced stress to the endoplasmic reticulum of mouse osteoblasts

Activating transcription factor 4 (ATF4) protein has a dual role in osteoblasts. It functions as a responder to stress to the endoplasmic reticulum (ER) as well as a transcription factor for bone formation. Little is known about molecular pathways that can potentially lead to stress-induced apoptosis or homeostasis of extracellular matrix (ECM) molecules. Based on microarray-derived mRNA expression data for mouse osteoblasts (MC3T3 E1 cells, clone 4), we analyzed the ER-stress responses in the presence of 10 nM Thapsigargin using two computational approaches: "Gene Set Enrichment Analysis (GSEA)" and "Ingenuity Pathways Analysis (IPA)." GSEA presented a strong linkage to an expression pattern observed in the responses to hypoxia, and IPA identified two molecular pathways: ATF4-unlinked connective tissue development and ATF4-linked organ morphology. Real-time polymerase chain reaction (PCR) and Western blot analyses validated eIF2alpha-driven translational regulation as well as ATF4-linked transcriptional activation of transcription factors and growth factors including FOS, FGF-9, and BMP-2. Consistent with the role of p38 MAPK in hypoxia, phosphorylation of p38 MAPK was activated in nonapoptotic osteoblasts under surviving ER stress. Furthermore, the level of phosphorylated PERK was elevated. These results support cross-talk between p38 MAPK and ER kinase, presenting a similarity to the responses to hypoxia as well as a pathway toward connective tissue development and organ morphology.

Topological Analysis of MAPK Cascade for Kinetic ErbB Signaling

Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules.

Synthetic double-stranded RNA induces retinoic acid-inducible gene-I in mouse osteoblastic cells

Retinoic acid inducible gene-I (RIG-I) is a member of the DExH box family of proteins. RIG-I acts as a sensor of viral infections through the recognition of viral double-stranded RNA (dsRNA). Recently, it was demonstrated that polyinosinic acid:polycytidylic acid [poly(I):poly(C)], a synthetic dsRNA analogue, induced the expression of RIG-I in various cell types, such as vascular endothelial cells and gingival fibroblasts. However, it remains unclear whether RIG-I is induced in osteoblasts in response to poly(I):poly(C). In the present study, we investigated the effects of poly(I):poly(C) on the expression of RIG-I in mouse osteoblastic MC3T3-E1(E1) cells. We found that poly(I):poly(C) increased the expression level of RIG-I in E1 cells, and that recombinant interferon-β (IFN-β) induced the expression of RIG-I mRNA in E1 cells. An anti-IFN-β neu-tralizing antibody partially inhibited poly(I):poly(C)-induced RIG-I expression. These results indicate that RIG-I production is induced by poly(I):poly(C)-provoked IFN-β in mouse osteoblastic E1 cells. We suggest that osteoblasts are involved in antiviral defense as well as in bone metabolism. Results of further studies will provide more clues regarding the molecular function of osteoblasts in viral infection.

Toll-like receptor 3 ligand-induced antiviral response in mouse osteoblastic cells

Double-stranded RNA (dsRNA) and its mimic, polyinosinic acid:polycytidylic acid [poly(I):poly(C)], are recognized by toll-like receptor 3 (TLR3) that induces the production of IFN-beta in many cell types. In the present study, we investigated the effects of poly(I):poly(C) on mouse osteoblastic MC3T3-E1 (E1) cells. Poly(I):poly(C) markedly increased IFN-beta mRNA level in a dose-dependent manner. The increase in the IFN-beta mRNA level was apparent as early as 1 h after adding poly(I):poly(C) to the culture and peaked at 12 h. Stimulation with poly(I):poly(C) enhanced the expression of CXCL10 mRNA and TLR3 in E1 cells. Moreover, poly(I):poly(C) induced tyrosine phosphorylation of the transcription factor STAT1 in E1 cells. An anti-IFN-beta neutralizing antibody partially inhibited poly(I):poly(C)-induced CXCL10 mRNA, TLR3 mRNA and STAT1 phosphorylation. These results indicate that osteoblasts secrete IFN-beta in response to viral infection and that endogenous IFN-beta induces both CXCL10 and TLR3 production via an IFN-alpha/beta receptor-STAT1 pathway. It is suggested that osteoblasts are involved in host defense as well as bone metabolism.

Synthetic LXXLL peptide antagonize 1,25-dihydroxyvitamin D3-dependent transcription.

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) through its ability to regulate cellular programs of gene expression. We identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides using a mammalian two-hybrid system and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. Peptides were identified that were reactive to RXR alone as well as to both VDR and RXR. Peptide fusion proteins were then examined in MC3T3 E1 cells for their ability to block induction of the osteocalcin promoter by 1,25(OH)(2)D(3) or stimulation of an RARE-TK reporter by 9-cis RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)(2)D(3)-dependent transcription by up to 75%. Peptides that interacted with RXR blocked 9-cis RA induced transcription. Two RXR-interacting peptides, however, were also found to block 1,25(OH)(2)D(3) response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)(2)D(3)-induced osteocalcin gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)(2)D(3).

Histamine stimulates production of osteoclast differentiation factor/receptor activator of nuclear factor-κB ligand by osteoblasts

Histamine H 1, H 2 , and H 3 receptors are expressed by osteoblastic MC3T3-E1 (E1) cells derived from mouse calvaria. Expression of the osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-κB ligand (RANKL) transcript was induced in E1 cells and bone marrow stromal cells (ST2). Histamine markedly increased the steady-state level of ODF/RANKL mRNA in a dose-dependent manner. The effect of histamine on expression of ODF/RANKL mRNA by E1 cells was transient, with a peak at 6 h. Western blot analysis revealed that histamine increased production of ODF/RANKL protein by E1 cells at 12 h. In cocultures of E1 cells and mouse bone marrow cells, histamine stimulated osteoclastogenesis in the presence of 1,25-dihydroxyvitamin D 3 and this effect was blocked by preincubation with neutralizing antibody against ODF/RANKL. These results suggest that histamine regulates osteoclastogenesis, at least in part, through induction of ODF/RANKL expression by osteoblasts and bone marrow stromal cells.

Human monocytic cell lines transformed in vitro by Epstein-Barr virus display a type II latency and LMP-1-dependent proliferation.

Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fms gene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of the EBNA-1 and LMP-1, but not EBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to an LMP-1 antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.

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.

Low Calcium Environment Effects Osteoprotegerin Ligand/Osteoclast Differentiation Factor

In coculture with osteoblastic cell line MC3T3-E1 (E1) and mouse bone marrow cells, we reported that numbers of osteoclasts rose significantly on exposure to a low-calcium environment. Here we examined how osteoblasts influence osteoclastogenesis under a low-calcium environment. Comparing low extracellular calcium with a regular calcium environment, osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) mRNA expression show more increase in the culture of low-calcium environment than in that of a regular calcium environment. Calcium-sensing receptor (CaSR), which was supposed as one of the mechanisms of recognizing extracellular calcium, existedon the surface of E1 cells. When E1 cells stimulated with agonists of CaSR, gadolinium, and neomycin, OPGL/ODF mRNA expression decreased. Moreover, these agonists reduced osteoclast formation in coculture. Taken together, it is possible that osteoblasts may recognize extracellular calcium via CaSR and regulate osteoclastogenesis.

Effect of Extracellular Calcium Concentrations on Osteoclast Differentiation in Vitro

We have been researching with the purpose of understanding the hard organization mineralization-suppression mechanism under a low calcium environment. So far, we have discerned clearly that the femur of an incubated newborn rat under a low calcium environment will develop bone formation dyscrasia such as hypertrophy of the caput. In this experiment, MC3T3-E1 (E1) cells, which are osteoblast-like cells, and mouse bone marrow were incubated in a coculture system under a low calcium environment and manifestation of osteoblast cells and their ability to resorb were examined. The results suggested that as the calcium concentration in the medium decreased manifestation of osteoclast cells increased, and as the degree of mineralization of E1 cells advanced, the number of manifestation of osteoblast cells decreased. We, therefore, report that there is a possibility that extracellular calcium concentrations involve the process of differentiation of bone marrow cells into osteoblast cells.

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.

Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata).

Bacterial feeding nematodes in the order Rhabditida including Zeldia punctata (Cephalobidae) and Caenorhabditis elegans (Rhabditidae) differ profoundly in the buccal capsule parts and associated cells. We carried out a range of tests to determine which buccal capsule parts and cells are evolutionarily homologous between the representative species of the two families. Tests included reconstruction of the buccal capsule and procorpus with transmission electron microscopy (TEM), nuclei position and morphology using 4, 6-diamidino-2-phenylindole (DAPI) staining, and cell lineage using four dimensional (4D) microscopy. The lining of the buccal capsule of Z. punctata and additional Cephalobidae includes four sets of muscular radial cells, ma, mb, mc and md, in contrast to C. elegans and additional Rhabditidae, which has two sets of epithelial cells (e1, e3) and two sets of muscle cells (m1, m2). Cell lineage of a nematode closely related to Z. punctata, Cephalobus cubaensis, supports the hypothesis that in cephalobids the e1 and e3 cells become hypodermal cells or are programmed to die. Our findings contradict all previous hypotheses of buccal capsule homology, and suggest instead that ma and mb in Z. punctata are homologous to m1 and m2 in C. elegans respectively. We also hypothesize that ma and mb could be homologous to primary and secondary sets of stylet-protractor muscle cells in the plant parasitic Tylenchida.

Identification of a Transforming Growth Factor-β1/Bone Morphogenetic Protein 4 (TGF-β1/BMP4) Response Element within the Mouse Tissue Transglutaminase Gene Promoter

Tissue transglutaminase is a calcium-dependent, protein cross-linking enzyme that is highly expressed in cells undergoing apoptosis. The expression of tissue transglutaminase is regulated by a variety of molecules including retinoids, interleukin-6, and transforming growth factor-beta1 (TGF-beta1). Retinoid and interleukin-6 inductions of tissue transglutaminase expression are mediated by specific cis-regulatory elements located within the first 4.0 kilobase pairs of the promoter of the gene. The present studies were designed to identify the molecular mechanisms mediating the regulation of tissue transglutaminase gene expression by TGF-beta family members. Transient transfection of Mv1Lu cells with transglutaminase promoter constructs demonstrated that 0.2 nM TGF-beta1 maximally induced the activation of the promoter through a 10-base pair TGF-beta1 response element (TRE; GAGTTGGTGC) located 868 base pairs upstream of the transcription start site. This same element mediated an inhibitory activity of TGF-beta1 on the transglutaminase promoter in MC3T3 E1 cells. The TRE through which TGF-beta1-regulated the activity of the transglutaminase promoter was necessary and sufficient for bone morphogenetic protein 2- (BMP) and BMP4-dependent inhibition of the tissue transglutaminase promoter. The TGF-beta1, BMP2, and BMP4 regulation of the transglutaminase promoter activity was similar to the responses we observed for the endogenous transglutaminase activity of Mv1Lu and MC3T3 E1 cells. For BMP2 and BMP4, this regulation was paralleled by a decrease in tissue transglutaminase mRNA in MC3T3 E1 cells. The results of these experiments suggest that TGF-beta1, BMP2, and BMP4 regulation of mouse tissue transglutaminase gene expression requires a composite TRE located in the 5'-flanking DNA.

Human alpha 2-macroglobulin is an osteogenic growth peptide-binding protein.

The osteogenic growth peptide (OGP) is a 14mer mitogen of osteoblastic and fibroblastic cells. Physiologically, OGP is present in high abundance in human and other mammalian sera. Most of the serum OGP is complexed noncovalently to heat sensitive, high molecular weight OGP-binding proteins (OGPBPs). Changes in serum OGP levels that follow bone marrow ablation and the low doses of exogenous OGP required for the stimulation of bone formation suggest a regulatory role for the OGPBPs. In the present work, the OGP binding activity was monitored by competitive binding to [3-125I(Tyr10)]-sOGP and the corresponding complexes were demonstrated on nondenaturing cathodic polyacrylamide gel electrophoresis. We show that OGP binds to both native and activated human plasma alpha 2-macroglobulin (alpha 2M). alpha 2M was also immunoidentified in reduced and nonreduced SDS-polyacrylamide gel electrophoresis of OGP-affinity purified plasma-derived proteins. Immunoreactive OGP was detected in commercial preparations of both forms of alpha 2M; OGP was purified to homogeneity from the commercial preparation of activated alpha 2M. In MC3T3 E1 cells, native alpha 2M, at concentrations < 50 ng/mL, had a substantially increased mitogenic effect in the presence of synthetic, native-like, OGP (sOGP). Similar amounts of activated alpha 2M inhibited the sOGP proliferative effect. These results suggest that the native alpha 2M enhances the immediate availability of OGP to its target cells. Activated alpha 2M may participate in the removal of OGP from the system.

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals in increases osteogenesis and hemopoiesis. In stromal cell cultures OGP stimulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP in high abundance is present in normal human and animal serum mainly complexed to OGP binding protein (OGPBP) or proteins. Here we show the presence of two OGPBPs, OGPBP-1, and OGPBP-2, in cultures of osteoblastic MC3T3 E1 cells. Immunoreactive OGP (irOGP) also accumulates in the medium of these cultures and in cultures of NIH 3T3 fibroblasts. A large amount of irOGP was released by heat inactivation of OGPBP-2 and purified by ultrafiltration and hydrophobic HPLC. The purified irOGP was identical to OGP obtained previously from rat regenerating bone marrow and human serum in terms of its amino acid sequence, immunoreactivity, and mitogenicity. Osteoblastic and fibroblastic cell proliferation can be arrested by anti-OGP antibodies and rescued by exogenous OGP, indicating that in the absence of serum or other exogenous growth stimulators the endogenously produced OGP is both necessary and sufficient for baseline proliferation. The OGP production is up- and down-regulated, respectively, by low and high doses of exogenous OGP in a manner consistent with an autoregulated feedback mechanism. The most effective OGP dose in MC3T3 E1 cells is at least two orders of magnitude lower than that in non-osteoblastic cell systems. This differential sensitivity of the osteoblastic cells could result in a preferential anabolic effect of OGP in bone.

Mitogenic action of osteogenic growth peptide (OGP) Role of amino and carboxy-terminal regions and charge

We have recently reported the discovery of a 14-amino-acid osteogenic growth peptide (OGP). In vivo OGP increases bone formation and trabecular bone density. Physiologically it is found in serum complexed to an OGP binding protein (OGPBP). In vitro OGP has a biphasic effect on osteoblastic MC 3T3 E1 and fibroblastic NIH 3T3 cell proliferation; at low concentrations (0.01–1.0 and 1.0–100.0 pM, respectively) it is highly stimulatory with an inhibition at higher doses. To assess possibilities of labeling synthetic OGP to obtain radio- or fluorescent ligands, OGP analogues were extended at the N- or C-termini with Cys or Cys(S-NEtSucc) or the OGP Tyr-10 replaced by 3-I(Tyr). All analogues with N-terminal modifications, as well as the [Cys 15]OGP-NH 2 retained the OGP-like dose-dependent effect on proliferation of the MC 3T3 E1 and NIH 3T3 cells, although the magnitude of stimulation was lower, approx. 2 3 that of the native-like synthetic OGP. The [Cys 15(S-NEtSucc)]OGP-NH 2 and [3-I(Tyr 10)]OGP shared only the inhibitory activity of OGP. This suppression is further shared by a number of other positively and negatively net charged, but not net neutral, peptides. Both N-terminal-modified analogues displayed a decreased binding activity to the OGPBP. All analogues except reverse OGP, [3-I(Tyr 10)]OGP and [Cys 15(S-NEtSucc)]OGP-NH 2 reacted with anti-OGP antibodies. These data are not only important for labeling purposes but suggest a respective role for the OGP N-and C-terminal regions in binding to the OGPBP and putative OGP receptor. It appears that the OGP proliferative activity represents the net effect of stimulation specific to the OGP structure and nonspecific inhibition associated with the peptide's high positive net charge.