Conditioned Media Samples Research Articles

Substance P stimulates release of RANKL via COX-2 expression in human dental pulp cells

Our previous study found that substance P (SP), a sensory neuropeptide, was expressed in the dental pulp of rats during experimental tooth movement. We examined the effects of SP on the production of prostaglandin (PG) E2 and the receptor activator of nuclear factor- B ligand (RANKL) by human dental pulp fi broblast-like (HDPF) cells. SP was added to cultured HDPF cells at concentrations ranging from of 10(-4) to 10(-12) mol/L. PGE2 and soluble RANKL (sRANKL) levels were determined using enzyme-linked immunosorbent assay kits. Gene expression was confi rmed by RT-PCR analysis. Pit formation assays using dentin slices were carried out to examine the effect of SP on osteoclastogenesis. The levels of PGE2 and sRANKL increased in the presence of SP, though the increases were greater in the experimental groups in both a time- and concentration-dependent manner, and the increase of RANKL was partially mediated by PGE2. The gene expression of cyclooxygenase (COX)-2 and RANKL was up-regulated, and conditioned medium samples obtained from HDPF cells treated with SP induced bone resorption. SP stimulated the production of PGE2 and RANKL, and promoted bone resorption. Therefore, SP may be involved in pulpal inflammation and root resorption during orthodontic tooth movement.

A ROLE FOR TGF?? IN CONTROLLING HUMAN ISLET PLASTICITY

The plasticity of pancreatic cells is well documented in the context of both regeneration and carcinogenesis, and extends to the cells of the islet. The regenerative capacity of the pancreas is such that upon 90% pancreatectomy, the organ regains 45% of its lost endocrine cell mass within 8 weeks. However, cell types that exhibit plasticity are also subject to increased rates of carcinogenesis. TGFβ has been implicated in pancreatic regeneration and loss of TGFβ signaling is common in pancreatic cancer. Human islet-enriched fractions (>70% purity) were embedded in a type 1 collagen matrix and cultured in serum-free DMEM/F12 with 1 μM dexamethasone, 10 ng/ml EGF, 24 mU/ml insulin and 200 ng/ml cholera toxin. Over the course of 8 days, islets transformed into duct-like structures (DLS) in a process characterized by β-cell apoptosis and mantle cell transdifferentiation. These DLS express markers of pancreatic ductal epithelium as well as endocrine progenitors. These culture conditions caused 55.2 ± 4.4% (mean ± SEM) of islets to form DLS. Addition of TGFβ1 to the culture medium caused a dose-dependent decrease in DLS formation, with 10, 100 and 1000 pM TGFβ1 limiting DLS formation to 74.6 ± 2.9, 28.2 ± 5.8 and 6.2 ± 1.8% of control (p < 0.001 vs. control). Conversely, addition of neutralizing antibody (10 μg/ml) increased DLS formation rates to 129.6 ± 6.1% of control (p < 0.02), suggesting endogenous TGFβ production in islet cultures. Analysis of conditioned media samples detected as much as 0.35 ± 0.08 pM TGFβ1/2000IE·day, peaking in the first 2 days of culture and declining thereafter. Inhibition studies determined that the effects of TGFβ1 were mediated by the TGFβRI kinase and were p38 dependent. While TGFβRI and II and SMAD2 were detected in β-cells within the islet, suggesting inhibition of β-cell apoptosis as a possible mechanism of action, DLS cells expressed higher levels of these TGFβ signaling molecules. Thus, TGFβ appears to play a role in determining islet phenotype. Based on the TGFβ response, the islet-to-DLS model may be of use to study pancreatic carcinogenesis and regeneration.

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

5-Lipoxygenase regulates senescence-like growth arrest by promoting ROS-dependent p53 activation

5-Lipoxygenase (5LO) is involved in the production of leukotrienes and reactive oxygen species (ROS) from arachidonic acid. Its strong activation has been associated with several diseases like cancer and neurodegeneration. Here we show that 5LO activity increases during senescence-like growth arrest induced by oncogenic ras or culture history in both human and mouse embryo fibroblasts. Overexpression of 5LO promotes senescence-like growth arrest via a p53/p21-dependent pathway, and this occurs independently of telomerase activity. 5LO stabilizes p53 through phosphorylation at Ser15 and increases expression of the p53-transcriptional target p21. This is achieved by regulating ROS production. Indeed, ROS are increased in 5LO-arrested cells. Antioxidants and a low oxygen environment prevent 5LO-induced growth arrest. Finally, 5LO inhibition reduces the growth arrest induced by oncogenic ras or culture history and these effects are neutralized by the addition of exogenous ROS. These data link the 5LO pathway to oxidative crises of primary fibroblast and suggest that the ability of 5LO to induce senescence-like growth arrest may be important in the pathogenesis of 5LO-associated disorders.

High-Level Expression of Porcine Factor VIII from Murine Mesenchymal Stem Cells.

Low-level expression of human factor VIII (fVIII) has limited the success of clinical trials for hemophilia A using both ex vivo and in vivo gene transfer methods. Ex vivo genetic modification provides increased control of gene transfer and permits thorough characterization of transgene copy number, chromosomal integration site(s), and expression levels prior to transplantation. A subpopulation of adherent bone marrow-derived cell types, termed mesenchymal stem cells (MSCs), comprise an attractive target cell type for ex vivo gene transfer due to their accessibility, ability to differentiate into multiple cell types, and long-term survival following transplantation. Recently we demonstrated, in vitro, that recombinant B-domain-deleted porcine fVIII (rp-fVIII) is expressed at levels 10 – 14-fold greater than recombinant B-domain-deleted human fVIII (rh-fVIII) due to an enhanced rate of secretion from baby hamster kidney-derived (BHK-M) cells. Additionally we found that only the A1 and activation peptide-A3 domain sequences of porcine fVIII are necessary to retain high-level expression of hybrid human/porcine fVIII constructs. Here we report the expression of rp-fVIII from murine MSCs isolated from exon-16 fVIII knockout mice following ex vivo retroviral transduction. MSCs were transduced with ecotropic envelope-pseudotyped murine stem cell virus containing a rp-fVIIII transgene at an multiplicity of infection of 2 – 5 functional viral particles/target MSC. Following this transduction regimen the cell population harbored an average of 1.8 proviral genomes/cell as determined by quantitative real-time PCR. During culture in growth medium supplemented with 20% fetal bovine serum (FBS) rp-fVIII-transduced MSCs demonstrated a steady-state level of 2,400 fVIII mRNA transcripts/cell and an apparent fVIII production rate of 174 units/106 cells/24 hr as determined by quantitative real-time RT-PCR and one-stage clotting assay, respectively. However, when cultured in serum-free medium the mRNA levels decreased to 950 transcripts/cell and apparent fVIII production was reduced to 14 units/106 cells/24 hr. The latter mRNA/fVIII expression ratio is in agreement with that previously reported from stably transduced BHK-M clonal cell lines. In contrast the former mRNA levels are lower than predicted from the observed fVIII activity levels. The activation quotient (ratio of apparent fVIII activity following thrombin pre-treatment to non-thrombin treated baseline fVIII activity) increased 17-fold when the cells were cultured in serum-free versus serum-containing medium. Additionally, SDS-PAGE analysis of immunoprecipitated fVIII protein from serum-containing and serum-free medium revealed the presence of activated fVIII (fVIIIa) in conditioned medium samples containing FBS. Highly purified rp-fVIII from transduced-MSCs cultured in serum-free medium displayed similar relative mobility to BHK-M produced rp-fVIII upon SDS-PAGE analysis. These data warn against the determination of fVIII activity in serum-containing medium using the one-stage coagulation assay due to the presence of activated fVIII with high specific activity. Based on these results it is reasonable to predict that hemophilia A could be cured using high-level expression fVIII constructs by transplantation of a feasible number (106 - 108) of cells containing a single copy of rp-fVIII or other high-level expression hybrid human/porcine fVIII transgenes.

159EFFECT OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) AND SERUM, ALONE OR IN COMBINATION, ON INTERFERON-TAU (IFNT) PRODUCTION BY OVINE EMBRYOS

The objectives were to establish whether GM-CSF, which may stimulate IFNT production by blastocysts at implantation, influences IFNT production by ovine embryos in culture and whether GM-CSF can explain stimulation of IFNT production by ovine serum (Rooke et al., 2003 Reprod. Abstr. Series 30:56). Oocytes, aspirated from sheep ovaries obtained from a local abattoir on 4 separate days, were matured and fertilized by standard procedures. On Day 1, cleaved zygotes (approximately 10 per 0.05-mL−1 drop; total, 175 per treatment) were cultured in synthetic oviductal fluid containing either 0.3% bovine serum albumin and amino acids (SOFA) or 10% sheep serum (SOFS) in the presence (+) or absence (−) of 5ngmL−1 ovine GM-CSF. Medium was changed every 2 days. On Days 6 and 7, blastocysts were removed from their group drops and assigned in a balanced manner individually to 0.05-mL−1 drops of one of the 4 media for a further 24h. Embryo grade, diameter and cell number were recorded. IFNT concentrations in conditioned media samples were determined by ELISA. The presence of serum accelerated blastocyst development (cross-tabulation; chi-square analysis; P=0.039; Table 1). Both serum and GM-CSF in group culture media increased (2×2 factorial ANOVA, P&amp;lt;0.001) IFNT concentrations (ngmL−1; SOFA−, 0.5; SOFA+, 1.7; SOFS−, 2.7; SOFS+, 4.2; SED, 0.47) with no interaction between serum and GM-CSF. After 24h individual culture of Day 6 blastocysts, IFNT concentrations (2×2 factorial ANOVA; Table 1) were influenced mainly by the origin of the blastocyst (group treatment covariate, serum, P&amp;lt;0.001; GM-CSF, P&amp;lt;0.001) rather than by individual blastocyst culture media (GM-CSF, NS; serum, P&amp;lt;0.001). The pattern of results was similar for Day 7 blastocysts. Incubation of zygotes in the presence of GM-CSF had no effect on embryo grade or diameter but blastocysts (both day 6 and 7) incubated individually for 24h in the presence of GM-CSF had more cells (128 v. 119; SED, 3.52; P=0.001) than blastocysts incubated concurrently without GM-CSF. However, the pattern of IFNT production after correction for cell number was unchanged. In conclusion, culture of zygotes in medium containing GM-CSF or serum, alone or in combination, from Days 1 to 7 increased IFNT production, and IFNT production by individually cultured blastocysts depended on their origin. SAC receives financial support from the Scottish Executive Environment and Rural Affairs Department. Table 1 Blastocyst yields (% of zygotes) on Days 6 and 7 and IFNT concentrations (ngML−1) in media after individual culture of Day 6 blastocysts

Interleukin-10 levels in single embryo conditioned media samples help predict implantation

Objectives: Preimplantation embryos secrete several different cytokines. T helper 2 class (Th2) cytokines promote humoral immunity and are implicated in pregnancy maintenance. Specifically, the Th2 cytokine, IL-10, may be important in modulating immune responses at the maternal-fetal interface. Here, we evaluate whether the presence or absence of IL-10 in culture media isolated from single embryos is associated with human embryonic implantation.

Proteins produced by barley microspores and their derived androgenic structures promote in vitro zygotic maize embryo formation

Maize zygotes formed in planta were isolated and co-cultured with barley microspores. Evidence suggests that culture with microspores and/or conditioned media in barley promoted zygotic embryo formation. In order to characterise active substances present in the conditioned media, we collected medium at various times after the initiation of culture. We showed that proteins appeared over time and that their quantity increased during the course of the culture. Some proteins were glycosylated as revealed by the ConA peroxidase test. The use of the antigen β-glucosyl Yariv reagent has shown that arabinogalactan-proteins (AGPs) were present. In addition, conditioned medium samples were analysed for their oligosaccharide content. New oligosaccharides appear in the course of the culture but they do not seem to affect development. We discuss in details the results in the context of understanding cell-cell interactions between embryo and nurse cells and the possible parallel with that occurs in ovulo between endosperm and embryo.

Human placental cytotrophoblasts attract monocytes and CD56(bright) natural killer cells via the actions of monocyte inflammatory protein 1alpha.

During human pregnancy, the specialized epithelial cells of the placenta (cytotrophoblasts) come into direct contact with immune cells in several locations. In the fetal compartment of the placenta, cytotrophoblast stem cells lie adjacent to macrophages (Hofbauer cells) that reside within the chorionic villus stroma. At sites of placental attachment to the mother, invasive cytotrophoblasts encounter specialized maternal natural killer (NK) cells (CD56(bright)), macrophages, and T cells that accumulate within the uterine wall during pregnancy. Here we tested the hypothesis that fetal cytotrophoblasts can direct the migration of these maternal immune cells. First, we assayed the chemotactic activity of cytotrophoblast conditioned medium samples, using human peripheral blood mononuclear cells as targets. The placental samples preferentially attracted NK cells (both CD56(dim) and CD56(bright)), monocytes, and T cells, suggesting that our hypothesis was correct. A screen to identify chemokine activity through the induction of a Ca(2)+ flux in cells transfected with individual chemokine receptors suggested that cytotrophoblasts secreted monocyte inflammatory protein (MIP)-1alpha. This was confirmed by localizing the corresponding mRNA and protein, both in vitro and in vivo. MIP-1alpha protein in conditioned medium was further characterized by immunoblotting and enzyme-linked immunosorbent assay. Immunodepletion of MIP-1alpha from cytotrophoblast conditioned medium showed that this chemokine was responsible for a significant portion of the induced monocyte and CD56(bright) NK cell chemotaxis. These data suggest the specific conclusion that cytotrophoblasts can attract monocytes and CD56(bright) NK cells by producing MIP-1alpha and the more general hypothesis that these cells may organize and act on leukocytes at the maternal-fetal interface.

17-beta estradiol elicits an autocrine leiomyoma cell proliferation: evidence for a stimulation of protein kinase-dependent pathway.

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2-dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP-kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2-stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI-3-K, and PLCgamma, and the concomitant activation of ancillary protein kinases, are related to E2-induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti-PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2-induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down-regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation.

Stromelysin-3 Is Induced in Tumor/Stroma Cocultures and Inactivated via a Tumor-specific and Basic Fibroblast Growth Factor-dependent Mechanism

Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) with a unique pattern of expression and substrate specificity. Unlike other MMPs, STR-3 is consistently and dramatically overexpressed by multiple epithelial malignancies, including carcinomas of the breast, lung, colon, head and neck, and skin. Recent studies suggest that STR-3 promotes the local establishment of epithelial malignancies, contributing to tumor cell survival and implantation in host tissues; however, STR-3's mechanism of action remains undefined. STR-3 is a stromal cell product, prompting speculation that infiltrating stromal cells secrete STR-3 in response to tumor-derived factors. To explore this possibility, we developed a tumor/"stroma" coculture assay in which non-small cell lung cancer (NSCLC) cell lines were grown on confluent monolayers of normal pulmonary fibroblasts. In these tumor/stroma cocultures, NSCLCs stimulate normal pulmonary fibroblasts to secrete STR-3 and release extracellular basic fibroblast growth factor. Thereafter, STR-3 is processed at a unique internal sequence via a basic fibroblast growth factor- and MMP-dependent mechanism to a previously unidentified 35-kDa protein that lacks enzymatic activity. 35-kDa STR-3 is the most abundant STR-3 protein in tumor/stroma cocultures and is only detected when normal pulmonary fibroblasts are cultured with malignant bronchial epithelial cells. Therefore, the tumor-specific processing of STR-3 to the 35-kDa protein is likely to be an important regulatory mechanism.

The development and utility of a defined muscle and fat co-culture system

The utility of co-culture systems in defining the interactions between two different cell types has been well documented in the literature but is a relatively new tool for use in the study of cells derived from normal muscle tissue from meat animals. The majority of myonuclei in postnatal skeletal muscle cells (myofibers) result from satellite cell proliferation, differentiation and fusion with existing myofibers. As such, satellite cell culture systems that mimic postnatal myofibers have great potential for delineating the process of growth and repair of muscle mass. Other ways to simulate the environment of postnatal myofibers might include the development of co-culture systems using satellite cells, or satellite cell-derived myotubes, and other mesodermal cells commonly found associated with muscle tissue in vivo. This brief review describes our initial efforts to develop a defined satellite cell and preadipocyte co-culture system. We provide useful information about defined media requirements and requirements for proper cell orientation and growth on two different growth planes. We present summary data to suggest that differences were found between members of the insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) families of polypeptides, when conditioned media samples were analyzed from co-cultures composed of 3T3-L1 preadipocytes and satellite cells (with different propensities to undergo morphological fusion to form multinucleated myotubes). We also provide information about potential problems to avoid when initiating and conducting co-culture experiments. Such a co-culture system has application in the study of human obesity and also in the regulation of fat deposition in meat animals.

Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo.

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.

Cell type-specific inhibition of keratinocyte collagenase-1 expression by basic fibroblast growth factor and keratinocyte growth factor. A common receptor pathway.

Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner.

Bovine oviductal and embryonic insulin-like growth factor binding proteins: possible regulators of "embryotrophic" insulin-like growth factor circuits.

Bovine oviductal monolayer and vesicle primary cultures express insulin-like growth factor (IGF)-I and -II mRNAs and polypeptides. Early bovine embryos also express IGF-I, IGF-II, IGF-I receptor, IGF-II receptor, and insulin receptor mRNAs. This study reports the expression of IGF binding protein (IGFBP) mRNAs and polypeptides in bovine oviduct primary cultures and IGFBP mRNAs in preattachment embryos. Release of immunoreactive IGF-I and IGF-II by oviduct cultures and bovine blastocysts was also determined. IGFBP-2, -3, -4, and -5 transcripts were observed in oviduct primary cultures throughout an 8-day interval. IGFBP-1 and -6 mRNAs were consistently not detected in the oviduct. Messenger RNAs encoding IGFBPs -2, -3, and -4 were detected throughout bovine preattachment development, while transcripts encoding IGFBP-5 were detected only in blastocysts. IGFBP-1 and -6 transcripts were not detected in early embryos. Ligand blot analysis with 125I-labeled IGF-II revealed the presence of four prominent polypeptide bands of approximate molecular masses 24, 31, and 36 kDa, and a broad band extending from 46 to 53 kDa, in conditioned media samples prepared from oviduct primary cultures. Western immunoblot analysis confirmed the identity of the 24-kDa, 31-kDa, and 36-kDa species as IGFBP-4, -5, and -2, respectively. Levels of the release of IGF-II from oviductal vesicle cultures were significantly greater than levels observed for monolayer cultures (p < 0.005). No significant difference in the levels of IGF-I release between monolayer and vesicle cultures was observed. Pools of 10 blastocysts released on average 36.2 +/- 3.9 pg of IGF-II per embryo, while the release of embryonic IGF-I was below the levels of detection for our assay. The results suggest that maternally derived IGF may be regulated by IGFBPs to support bovine preattachment development.

A simplified method of analysis of cell-conditioned medium forInsulin-like Growth Factor-I (IGF-I) activity

To facilitate further investigation of the relationship between IGF-I and IGF binding proteins (IGFBPs) and their effect on myogenic satellite cells, we developed a procedure for rapid screening of cell-conditioned medium for free IGF-I. This method involves the adaptation and implementation of a DNA vacuum blotting manifold, which allows detection of a broad range of IGF-I and is rather sensitive (detection < 10 ng) when compared to western immunoblots. Additionally, this dot-blot method requires less than 40 minutes prior to the addition of the blotto-antibody mixture. Application of the dot-blot method for analysis of free IGF-I levels in conditioned medium samples offers an alternative to the use of western immunoblots, and may be used in initial screening of large numbers of conditioned medium samples.

Rabbit slow and fast skeletal muscle‐derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin‐like growth factor system

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37–40, 32, 30–31, 28, and 24 kDa. The 30–31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM-conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an ∼400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the α2β2 form of the IGF-I receptor, and two β subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two β subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two β subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system. © 1996 Wiley-Liss, Inc.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37-40, 32, 30-31, 28, and 24 kDa. The 30-31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an approximately 400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the alpha 2 beta 2 form of the IGF-I receptor, and two beta subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two beta subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two beta subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist.

Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 fibroblasts. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis.

Synthesis and release of 9-cis retinoic acid by the urodele wound epidermis

ABSTRACT The wound epidermis is a transient secretory epithelium that apposes the mesenchymal blastema of a regenerating urodele limb, and is required for regeneration. Previous studies have shown that the positional identity of the blastema is respecified by retinoic acid (RA; Maden, M. (1982)Nature 295, 672-675), that the blastema contains RA (Scadding, S. R. and Maden, M. (1994) Dev. Biol. 162, 608617), and that an RA-reporter gene introduced into the blastema is differentially activated along the proximodistal axis (Brockes, J. P. (1992)Proc. Natl. Acad. Sci. USA 89, 11386-11390). The newt limb wound epidermis has been explanted with minimal mesenchymal contamination and cultured under conditions where it retains expression and inducibility of marker antigens. We have assayed for the release of retinoids from the wound epidermis by coculture with cells transfected with an RA-responsive reporter gene. The reporter was activated to a level corresponding to stimulation by 0.1–1 nM RA, and this activation was substantially conferred by medium conditioned by the wound epidermis. No significant activation was observed for cells transfected with mutated reporter plasmids and analysed in parallel co-cultures. Wound epidermis from contralateral proximal and distal blastemas were compared for reporter activation, and gave a P/D activation ratio significantly greater than 1.Wound epidermis explants were cultured in the presence of tritiated retinol, and extracts were analysed by HPLC on three different columns. Radioactivity was detected in peaks corresponding to didehydroretinol, 9-cis RA and other unidentified metabolites. Analysis of conditioned media samples, some after pulse chase experiments, detected significant release of retinol, 9-cis RA and other metabolites. Although all-trans RA was detectable, the predominant acidic metabolite was 9-cis RA. These experiments establish the wound epidermis as a source of RA for local cellular interactions in the blastema.