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PubMed2005

Chemosensitization by STI571 targeting the platelet-derived growth factor/platelet-derived growth factor receptor-signaling pathway in the tumor progression and angiogenesis of gastric carcinoma.

BACKGROUND: Autocrine and paracrine growth mediated by the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR)-signaling pathway plays an important role in the progression of solid tumors. The authors assessed the effect of STI571 on the tumor growth of gastric carcinoma in combination with 5-fluorouracil (5-FU) or paclitaxel targeting the PDGF/PDGFR-signaling pathway. METHODS: In MKN-45 gastric carcinoma cells, the cytotoxic effect was evaluated by 3-(4,5 dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the in vivo antitumor effect was evaluated in a nude mouse xenograft. Both STI571 and an antitumor drug were administered intraperitoneally. Gene expression was assessed by Western blot analysis and immunohistochemical staining. Apoptotic cell death was evaluated by the terminal deoxyuridine triphosphate-biotin nick-end labeling assay, and tumor angiogenesis was evaluated by microvessel density analysis. RESULTS: Treatment with STI571 alone was not effective in vitro, as assessed by a 50% inhibitory concentration value of 24.3 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the cytotoxic effect somewhat when the concentration of STI571 was increased to 10 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the antitumor effect of the antitumor drug significantly in vivo. The enhanced antitumor effect was associated with increased apoptotic cell death and inhibition of tumor angiogenesis. Treatment with STI571 down-regulated the expression of PDGF-BB and PDGFR-beta in tumor cells and decreased the production of phosphorylated PDGFR-beta and phosphorylated Akt. Furthermore, treatment with STI571 inhibited the expression of PDGFR-beta in stromal cells. CONCLUSIONS: STI571 was an effective chemosensitizer of antitumor drugs, such as 5-FU and paclitaxel for gastric carcinoma, targeting the PDGF/PDGFR-signaling pathway of tumor cells and stromal cells in disease progression and angiogenesis.

Animals↗

PubMed2005

Biphasic calcium response of platelet-derived growth factor stimulated glioblastoma cells is a function of cell confluence.

BACKGROUND: Previous reports have linked the spiking or two-phased character of calcium transients evoked by platelet-derived growth factor (PDGF) to the position of cells in the cell cycle without regard to cell-cell contact and communication. Because cell confluence can regulate growth factor receptor expression and dephosphorylation, we investigated the effect of cell culture confluence and cell cycle on calcium responses of PDGF-BB-stimulated A172 glioblastoma cells. METHODS: Digital imaging cytometry was used to correlate the peak and duration of calcium response with bromodeoxyuridine positivity and DNA content and with culture confluence on a cell-by-cell basis. RESULTS: In serum-starved cultures, complete two-phase calcium signals and shorter, lower spikes occurred independent of cell cycle phase. However, the confluence of cell culture seemed essential for inducing a complete response because cells in sparse cultures exhibited mostly short spikes with lower peaks or no transients at all. CONCLUSION: Because cell confluence, by virtue of cell-cell contacts, is assumed to be an important regulator of proliferation, one is tempted to speculate that in transformed cells the ability to produce stronger growth signals upon reaching confluence and facing contact inhibition could provide a proliferative advantage.

Becaplermin↗

PubMed2005

Abnormal development of the intercostal muscles and the rib cage in Myf5-/- embryos leads to pulmonary hypoplasia.

The aim of our study was to investigate the importance of pulmonary distension and fetal breathing-like movements executed by the contractile activity of the intercostal respiratory muscles for proper lung growth and maturation. Lung development in Myf5-/- embryos, lacking the rib cage and functional intercostal musculature, was compared with wild-type controls at embryonic days 14.5, 16.5, and 18.5. Our data revealed that Myf5-/- embryos suffered from pulmonary hypoplasia in part due to the decreased number of proliferating lung cells and in part due to the increased number of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) -positive cells. In addition, the proximal-to-distal expression gradient of thyroid transcription factor-1 observed in wild-type embryos was not maintained in Myf5-/- embryos. The number of lung cells expressing platelet-derived growth factor-BB, its receptor and insulin growth factor-I was significantly decreased in the hypoplastic lung. By contrast, no difference in the expression pattern of surfactant associated proteins or Clara cells marker was detected between wild-type and Myf5-/- embryos. Collectively, our data suggest that the mechanochemical signal transduction pathway used in vitro is also effective in vivo influencing lung growth but not lung cell maturation and resulting in lung hypoplasia. These data affirm the role of fetal breathing-like movements in lung organogenesis.

Animals↗

PubMed2003

Activation of platelet-derived growth factor pathway in human asthmatic pulmonary-derived mesenchymal cells.

Cell cultures of mesenchymal type were obtained from biopsies taken after bronchoscopy from patients with asthma. It was possible to achieve outgrowth of fibroblast-like cells from these lung biopsies, which stained for alpha-smooth actin indicating that they were of myofibroblast type. Morphologically, two types of myofibroblasts could be observed: one intermediate form with more stretched cell shape and lamellipodia protrusions, and one more differentiated compact form of myofibroblast. The intermediate form was the most dominant type in these patients, indicating an active ongoing remodelling process. Further studies showed that platelet-derived growth factor (PDGF) might be the factor that stimulates the formation of the intermediate type of myofibroblasts, since it enhance migration of normal human lung fibroblasts 4-fold compared to control through an induced formation of stress fibers and lamellipodia protrusions. Additionally, intracellular signalling pathways involved in migration, such as RhoA and MAPkinase were stimulated 1.5-fold and 3.5-fold, respectively. By using two-dimensional (2-D) gel electrophoresis and protein identification by peptide mass finger printing matrix assisted laser desporption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) it was possible to confirm that PDGF affected the synthesis of proteins involved in the remodelling process, such as collagen VI and post-translational forms thereof. PDGF also stimulated the production of FK506 binding protein of 65 kDa, a protein involved in smooth muscle differentiation, and proteins involved in the rearrangement of the cytoskeleton connected to migration such as the actin related protein ARP3, the T-complex protein and the heat shock protein 60. We demonstrate that PDGF has a potential pathological role in asthma and formation of subepithelial fibrosis by inducing changes in the proteome.

Asthma↗

PubMed2002

Platelet-derived growth factor-b expression induced after rat peripheral nerve injuries.

Schwann cells are crucially important for peripheral nerve regeneration. These cells synthesize several factors that are supposed to enhance axonal regeneration when injured. Platelet-derived growth factor (PDGF) B-chain and its beta-receptor are expressed in Schwann cells in both normal peripheral nerves and culture. To elucidate the role of PDGF-B in peripheral nerve regeneration, we investigated its expression in cut or crush-injured rat sciatic nerves for up to 28 days. Northern blotting identified substantial increase of PDGF B-chain transcripts in injured nerves. Immunohistochemistry demonstrated that protein products of the transcripts were augmented at the distal tip of swollen axons in proximal nerve segments and in regenerating axons. Soon after both types of injury, considerable amounts of PDGF-B accumulated in numerous Schwann cells in distal segments of both models. With restoration of the axon-Schwann cell relationship in the crush model, levels of PDGF-B tended to decrease, eventually returning to normal. In the cut model in which the relationship cannot be restored, the PDGF-B was depleted to a very low level. The spatiotemporal correlation between PDGF-B and cell proliferation was very close throughout the study. These results differed strikingly from those of our previous study of rat optic nerve transection, in which PDGF-B was expressed only in a few recruited macrophages and glial cells. Augmented PDGF-B expression after sciatic nerve injury might contribute to peripheral nerve regeneration because PDGF-B is a mitogen and survival factor for Schwann cells and because it has trophic activity on neurons.

Animals↗

PubMed2005

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase-derived reactive oxygen species (ROS) in PDGF-induced HSC proliferation. The human HSC line, LI-90 cells, murine primary-cultured HSCs, and PDGF-BB were used in this study. We examined the mechanism of PDGF-BB-induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF-BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF-BB-induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF-BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen-activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase-derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF-BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK.

Animals↗

PubMed2006

STAT1 inhibits liver fibrosis in mice by inhibiting stellate cell proliferation and stimulating NK cell cytotoxicity.

Liver fibrosis, a common scarring response to chronic liver injury, is a precursor to cirrhosis and liver cancer. Here, we identified signal transducer and activator of transcription 1 (STAT1) as an important negative regulator in liver fibrosis. Our findings show that disruption of the STAT1 gene accelerated liver fibrosis and hepatic stellate cell (HSC) proliferation in an in vivo model of carbon tetrachloride (CCl4)-induced liver fibrosis. In vitro treatment with IFN-gamma inhibited proliferation and activation of wild-type HSCs, but not STAT1-/- HSCs. Moreover, compared to wild-type cells, cellular proliferation stimulated by serum or platelet-derived growth factor (PDGF) was enhanced and accelerated in STAT1-/- HSCs, which was partially mediated via elevated PDGF receptor beta expression on such cells. Polyinosinic-polycytidylic acid (poly I:C) or IFN-gamma treatment inhibited liver fibrosis in wild-type mice but not in STAT1-/- mice. Induction of NK cell killing of activated HSCs by poly I:C was attenuated in STAT1-/- mice compared to wild-type mice, which was likely due to reduced NKG2D and TRAIL expression on STAT1-/- NK cells. Finally, activation of TGF-beta/Smad3 signaling pathway was accelerated, whereas induction of Smad7 was diminished in the liver of STAT1-/- mice after CCl4 administration compared to wild-type mice. In conclusion, activation of STAT1 attenuates liver fibrosis through inhibition of HSC proliferation, attenuation of TGF-beta signaling, and stimulation of NK cell killing of activated HSCs. STAT1 could be a new therapeutic target for treating liver fibrosis.

Animals↗

PubMed1999

In vitro migratory potential of rat quiescent hepatic stellate cells and its augmentation by cell activation.

In liver injury, hepatic stellate cells are considered to depart from the sinusoidal wall and accumulate in the necrotic lesion through migration and proliferation. In this study, we investigated the migratory capacity of quiescent stellate cells in vitro and analyzed the relationship with proliferative response. Freshly isolated stellate cells that were seeded in the upper chamber of Cell Culture Insert (Becton Dickenson, Franklin Lakes, NJ) started to migrate to the lower chamber at 1 day and increased in migration index to 19% at 2 days. Cells in the lower chamber were stretched in shape with many lipid droplets and showed quiescent properties, i.e., negative expression of alpha-smooth muscle actin (alpha-SMA) or platelet-derived growth factor receptor-beta (PDGFR-beta). Migratory capacity in quiescent cells was also shown in the Matrigel-coated insert. Matrix metalloproteinase-2 (MMP-2) messenger RNA expression was low just after isolation, but was enhanced as migration became prominent. Migrating cells further showed higher proliferative activity than resting ones. The presence of PDGF/BB and Kupffer cells accelerated stellate cell migration by the chemotactic mechanism and concurrently augmented proliferation, whereas that of dexamethasone and interferon-gamma (IFN-gamma) attenuated migration as a result of general suppression effects. Compared with quiescent ones, alpha-SMA and PDGFR-beta-positive activated stellate cells obtained by 14-day culture exhibited more rapid and prominent migration, being regulated by mediators in a similar manner as described previously. These data indicate that quiescent stellate cells undergo migration, which is linked to proliferation and enhanced by PDGF/BB and Kupffer cells, suggesting the involvement of this function in the initial phase of development of postnecrotic fibrosis.

Actins↗

PubMed2000

Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor tyrosine kinase found in focal adhesions. FAK has been indicated as a point of convergence of other signaling pathways including platelet-derived growth factor (PDGF) receptors, and recently, FAK tyrosine phosphorylation has been shown to be stimulated by PDGF. In the present study we assessed the role of Ras as a possible intermediate protein regulating PDGF-induced FAK tyrosine phosphorylation in human hepatic stellate cells (HSCs), liver-specific pericytes primarily involved in the pathogenesis of liver fibrosis. For this purpose, cells were first subjected to retroviral-mediated gene transfer with a dominant-negative mutant of Ras (N17Ras). This resulted in a marked inhibition of PDGF-induced FAK tyrosine phosphorylation together with the expected reduction of PDGF-induced extracellular signal-regulated kinase activity (ERK). Afterward, the effects of pharmacological agents potentially affecting Ras isoprenylation were evaluated. PDGF-induced FAK tyrosine phosphorylation, ERK activity and intracellular calcium increase, as well as the biological effects of this growth factor, (i.e., mitogenesis and cell migration) were effectively blocked by GGTI-298, an inhibitor of geranylgeranyltransferase I. Inhibition of Ras processing obtained with FTI-277, an inhibitor of farnesyltransferase, resulted in detectable effects only at high doses. Taken together, these results establish that Ras operates as a protein-linking PDGF-beta receptor to FAK in human HSCs, and that signaling molecules requiring geranylgeranylation may also be involved in this process.

Alkyl and Aryl Transferases↗

PubMed2002

Human Ly-6 antigen E48 (Ly-6D) regulates important interaction parameters between endothelial cells and head-and-neck squamous carcinoma cells.

Selectin ligands are crucial components in the interaction between endothelial cells and extravasating cancer cells and, thus, play an important role in metastasis formation. Head-and-neck squamous cell carcinoma (HNSCC) variants expressing high levels of E48, a human Ly-6 protein (E48(hi)), expressed higher levels of the fucose-generating FX enzyme and of the fucosylated E-selectin ligand sLe(a) than cells expressing low levels of E48 (E48(lo)). Signaling through E48 upregulated expression levels of these molecules in HNSCC. In this work, we provide further evidence supporting the E48-FX-sLe(a) link by showing that FX antisense oligonucleotides reduced sLe(a) expression levels in HNSCC. We also show that E48 may be causally involved in regulating expression levels in HNSCC of 2 additional enzymes involved in the biosynthesis of sLe(a), namely, ST-30 and FucTIII. Also, selectin-mediated adhesion of E48(hi) variants to activated HUVECs was significantly higher than that of E48(lo) variants. Transfection experiments utilizing sense or antisense E48 cDNA indicated that E48 may be causally involved in this adhesion. Chemokines are involved in the extravasation process of tumor cells. The release of chemoattractants from HNSCC variants differing in E48 expression was therefore analyzed. HNSCC did not release any chemoattractants but induced the release of such factors from HUVECs. Supernatants from E48(hi) variants were significantly more efficient than E48(lo) cells at inducing the release of chemoattractants from HUVECs. Transfection experiments indicated that E48 may be causally involved in the induction of chemoattractant release from HUVECs. Angiogenesis is an important manifestation of cancer-endothelium interactions. We therefore assayed for the presence of angiogenic factors in culture supernatants of HNSCC. Supernatants from E48(lo) variants contained significantly higher amounts of PDGF than E48(hi) cells. Transfection experiments indicated that E48 may be causally involved. Taken together, our results suggest that E48 controls important interaction parameters between HNSCC and endothelial cells.

Antigens, Ly↗

PubMed2003

Expression profiles of angiogenic growth factors in squamous cell carcinomas of the head and neck.

Inhibition of angiogenesis by blocking angiogenic cytokines or their pathways has become a major target in experimental cancer therapies. This therapeutical approach requires a profound knowledge of growth factor profiles that contribute to tumor growth and progression. The respective knowledge is presently rather incomplete for head and neck squamous cell carcinomas (HNSCC). Therefore we studied expression of several angiogenic cytokines including VEGF, bFGF, PDGF-AB, PDGF-BB, G-CSF and GM-CSF in HNSCC in vivo and in vitro. In tumor tissues expression of all cytokines was observed albeit with marked differences concerning intensity and distribution pattern. Quantification of the cytokines in the supernatant of 15 tissue-corresponding HNSCC cultures revealed that VEGF, PDGF-AB and less frequently GM-CSF were secreted in high amounts of up to 13 ng/ml/10(6) cells. Twenty percent of the HNSCC cultures expressed only 1 cytokine in biologically active amounts, 60% 2 or 3 and 20% expressed the maximum of 4 cytokines simultaneously. Interestingly, we observed a distinct cytokine pattern: HNSCC cells secreting only 1 or 2 cytokines presented always with either VEGF and/or PDGF-AB, while G-CSF and GM-CSF were secreted primarily together with VEGF and PDGF-AB. The number of cytokines expressed by HNSCC cells correlated with the microvessel density of the original tumor and with the clinical outcome: tumors producing at least 3 cytokines revealed a significantly poorer patient prognosis. Our data indicate a major role for VEGF and PDGF-AB in HNSCC and that the additional secretion of G-CSF or GM-CSF might contribute to a poorer prognosis in patients suffering from these tumors.

Adult↗

PubMed1993

Ash/Grb-2, a SH2/SH3-containing protein, couples to signaling for mitogenesis and cytoskeletal reorganization by EGF and PDGF.

The Src homology (SH) region 2 binds to phosphorylated tyrosine residues and SH3 domains may interact with cytoskeletal molecules and GTPase-activating proteins for Rho/Rac proteins (the small GTP-binding proteins related to Ras). The recently cloned Ash/Grb-2 protein, a 25-28 kDa molecule composed entirely of SH2 and SH3 domains, is a mammalian homolog of the Caenorhabditis elegans Sem-5 protein, which communicates between a receptor protein tyrosine kinase and a Ras protein. In the present study the function of Ash/Grb-2 was investigated by microinjecting cells with an anti-Ash antibody. The antibody abolished both S phase entry and the reorganization of actin assembly to ruffle formation upon stimulation with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). On the other hand, anti-Ash antibody had no effect on S phase entry or actin stress fiber formation induced by either serum or lysophosphatidic acid. Since the induction of DNA synthesis, ruffle induction and stress fiber formation involve a function of Ras, Rac activation and Rho activation respectively, the findings strongly suggest that Ash plays a critical role in the signaling of both pathways downstream from growth factor receptors to Ras and Rac. Consistent with this, Ash co-precipitated with EGF receptor from EGF-stimulated cells. Other proteins of approximately 21, 29, 135 and 160 kDa were also detected in the anti-Ash antibody immunoprecipitates, suggesting a role of Ash as a linker molecule in signal transduction downstream of growth factor receptors.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Expression of monocyte chemoattractant protein 1 in human osteoblastic cells stimulated by proinflammatory mediators.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine superfamily of genes that induces chemotaxis of monocytes in inflammatory processes. The effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), platelet-derived growth factor (PDGF-BB), parathyroid hormone (PTH), and 1,25(OH)2D3 on MCP-1 expression in human osteoblastic cells were compared. Inflammatory or proinflammatory cytokines stimulated the production of MCP-1 in normal human osteoblastic cells as determined by RIA. The osteotrophic mediators PTH and 1,25(OH)2D3 and PDGF-BB had no effect on MCP-1 expression. In further studies, the steady-state mRNA and MCP-1 protein levels in two human osteoblastic cell lines, MG-63 and SaOS-2, were examined. MCP-1 expression at both the protein and mRNA levels was greatly increased by IL-1 beta and TNF-alpha. At the mRNA level, IL-1 beta and TNF-alpha strongly induced MCP-1 expression; TGF-beta and IL-6 induced MCP-1 but to a lesser extent. No significant changes in MCP-1 mRNA or MCP-1 protein secretion were observed when cells were treated with PDGF-BB, PTH, and 1,25(OH)2D3. When tested on preosteoclasts, MCP-1 was shown to have no effect on the formation of multinucleated, tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells.

Autoradiography↗

PubMed1996

The effect of systemically administered PDGF-BB on the rodent skeleton.

Platelet-derived growth factor (PDGF), an osteoblast mitogen, has been demonstrated to accelerate fracture healing and periodontal bone repair when applied locally in vivo. To explore whether PDGF could stimulate bone formation in intact bone, we administered it systemically to rats rendered acutely estrogen-deficient. Because PDGF may stimulate bone resorption in vitro, PDGF was administered with and without an antiresorptive agent (alendronate). All treatments were given by intravenous injection 3 times a week for 6 weeks. Spinal bone mineral density (BMD) decreased by 5% in the vehicle-treated ovariectomized (OVX) rats by the end of the study as determined by DXA. Treatment with PDGF prevented this bone loss and significantly (p < 0.05) increased the bone density in the spine (9%) and whole skeleton (5.8%). Combined treatment with PDGF and alendronate resulted in a greater increase at the spine (18%) and whole skeleton (12.8%) than either agent alone. Histomorphometric analysis demonstrated that treatment with PDGF increased the osteoblast number and osteoblast perimeter without consistent changes in osteoclast estimates. Biomechanical testing demonstrated that PDGF administration increased the vertebral body compressive strength and femoral shaft torsional stiffness and resulted in a trend for enhanced femoral head shearing strength. Coadministration of alendronate further increased these indices of bone strength. PDGF administration also caused premature closure of the growth plate, decreased body fat, and resulted in extraskeletal collagen deposition. We therefore demonstrate, for the first time, that systemic administration of PDGF can increase bone density and strength throughout the skeleton.

Absorptiometry, Photon↗

PubMed1996

Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro.

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-beta 1 (TGF-beta 1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-beta 1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-beta 1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-beta 1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-beta 1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.

Alkaline Phosphatase↗

PubMed2002

Functional receptor for platelet-derived growth factor in rat embryonic heart-derived myocytes: role of sequestered Ca2+ stores in receptor signaling and antagonism by arginine vasopressin.

Platelet-derived growth factor (PDGF) is established to function importantly in the growth, development, and function of most cardiovascular tissues. However, evidence that the factor participates directly in the growth and development of the mammalian myocardium is lacking. H9c2 rat embryonic ventricular myocytes were found to respond to PDGF-BB with a rapid mobilization of cell-associated Ca2+ and increased rates of protein synthesis, followed by markedly increased rates of DNA synthesis. PDGF acted as a full mitogen for these myocytes. Evidence is provided that documents the expression of classical PDGF-beta, but not PDGF-alpha, receptors in H9c2 cells. Scatchard analysis revealed the presence of 44,000 beta-receptors per myocyte. Cell shortening and clustering of plasmalemmal beta-receptors occurred within 30 min of exposure to PDGF-BB. Treatment was also associated with a transient increase in the rate of synthesis of GRP78/BiP, consistent with a transitory release of Ca2+ from the sarcoplasmic/endoplasmic reticulum [S(E)R]. Increased rates of protein synthesis at early times of PDGF treatment were additive with those occurring in response to arginine vasopressin, indicating different mechanisms of translational upregulation by these agents. The mitogenic effects of PDGF were delayed by vasopressin, which causes H9c2 myocytes to undergo hypertrophy while promoting the persistent depletion of S(E)R Ca2+ stores. In the presence of PDGF, vasopressin did not induce hypertrophy. As compared to untreated myocytes, DNA synthesis in PDGF-treated myocytes was optimized at lower extracellular Ca2+ concentrations and was significantly less sensitive to inhibition by ionomycin. H9c2 cells appear to provide a useful embryonic cardiomyocyte model in which to examine both PDGF-activated proliferative and vasopressin-activated hypertrophic events and the importance of transient vs. sustained Ca2+ release in these events.

Animals↗

PubMed2002

Role of mTOR in the degradation of IRS-1: regulation of PP2A activity.

We have investigated the role of PI 3-kinase and mTOR in the degradation of IRS-1 induced by insulin. Inhibition of mTOR with rapamycin resulted in approximately 50% inhibition of the insulin-induced degradation of IRS-1. In contrast, inhibition of PI-3 kinase, an upstream activator of mTOR, leads to a complete block of the insulin-induced degradation. Inhibition of either PI-3 kinase or mTOR prevented the mobility shift in IRS-1 in response to insulin, a shift that is caused by Ser/Thr phosphorylation. These results indicate that insulin stimulates PI 3-kinase-mediated degradation of IRS-1 via both mTOR-dependent and -independent pathways. Platelet-derived growth factor (PDGF) stimulation leads to a lower level of degradation, but significant phosphorylation of IRS-1. Both the degradation and phosphorylation of IRS-1 in response to PDGF are completely inhibited by rapamycin, suggesting that PDGF stimulates IRS-1 degradation principally via the mTOR-dependent pathway. Inhibition of the serine/threonine phosphatase PP2A with okadaic acid also induced the phosphorylation and degradation of IRS-1. IRS-1 phosphorylation and degradation in response to okadaic acid were not inhibited by rapamycin, suggesting that the action of mTOR in the degradation of IRS-1 results from inhibition of PP2A. Consistent with this, treatment of cells with rapamycin stimulated PP2A activity. While the role of mTOR in the phosphorylation of IRS-1 appears to proceed primarily through the regulation of PP2A, we also provide evidence that the regulation of p70S6 kinase phosphorylation requires the direct activity of mTOR.

3T3 Cells↗

PubMed2002

BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells.

For bone development, remodeling, and repair; the recruitment of mesenchymal progenitor cells (MPC) and their differentiation to osteoblasts is mandatory. The process of migration is believed to be regulated in part by growth factors stored within the bone matrix and released by bone resorption. In this study, primary human MPCs and to osteoblasts differentiated progenitor cells were examined for chemotaxis in response to human basic fibroblast growth factor (rhbFGF), human transforming growth factor beta 1 (rhTGF-beta1), human platelet derived growth factor bb (rhPDGF-bb), human bone morphogenetic protein-2 (rhBMP-2), and recombinant bone morphogenetic protein-4 of Xenopus laevis (rxBMP-4) from 0.001 to 1.0 ng/ml each. The results of migration were expressed as a chemotactic index (CI). Migration of primary human progenitor cells was stimulated by rhBMP-2, rxBMP-4, and rhPDGF-bb in a dose-dependent manner. The increase of CI was up to 3.5-fold for rhBMP-2, 3.6-fold for rxBMP-4, and up to 22-fold for rhPDGF-bb, whereas rhTGF-beta1 and rhbFGF did not stimulate cell migration in the concentration range tested. In contrast differentiated progenitor cells behave similar to primary human osteoblasts. RhBMP-2, rhPDGF-bb, and rhTGF-beta1 stimulated the migration from 2.2 to 2.4-fold each, while rxBMP-4 and rhbFGF reached only a CI of 1.7-1.6. The effect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary human MPC, including the change in response to growth factors after differentiation suggests a functional role for recruitment of MPCs during bone development and remodeling, as well as fracture healing.

Becaplermin↗