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At least 685 records · Page 38Linked to original sources
PubMed1994

PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors.

To delineate potential angiogenic roles of platelet-derived growth factor (PDGF), we have investigated PDGF and its receptors on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro (angiogenic endothelial cells). Initiation of cord/tube formation by angiogenic endothelial cells required bovine or human serum. Neutralization of PDGF-BB in human serum with a monoclonal anti-PDGF-BB antibody reduced cord/tube formation by 37 +/- 10%, whereas neutralizing anti-PDGF-AA and an IgG isotype-matched control antibody had no effect. DNA synthesis in response to PDGF-BB increased as the cords and tubes developed; furthermore, PDGF-BB induced the incorporation of BrdU in the nuclei of cells associated with these structures. PDGF beta-receptor (PDGF-beta) mRNA increased concomitantly with cord/tube formation, and PDGFR-beta were specifically localized by immunocytochemistry to developing and mature cords and tubes. However, PDGFR-beta transcripts and protein were undetectable in nonangiogenic endothelial cells, and PDGF alpha-receptor mRNA was not expressed in either endothelial cell strain. In contrast to nonangiogenic endothelial cells, angiogenic endothelial cells did not express the PDGF B-chain, the required ligand for the PDGFR-beta. We conclude that (a) PDGF-BB can contribute to angiogenesis in vitro, (b) PDGFR-beta are specific for cord/tube-forming endothelial cells and mediate endothelial proliferation and cord/tube formation, and (c) in angiogenic and nonangiogenic endothelial cells, the expression of PDGFR-beta and PDGF B-chain is inversely correlated. We therefore suggest that paracrine PDGF might amplify angiogenesis via direct action on endothelially expressed PDGFR-beta.

Animals↗
PubMed1995

The chemotactic response to PDGF-BB: evidence of a role for Ras.

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase-mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF-stimulated chemotaxis but may not be required for fibronectin-stimulated cell motility.

3T3 Cells↗
PubMed1996

Extracellular matrix alters PDGF regulation of fibroblast integrins.

Extracellular matrix (ECM) and growth factors are potent regulators of cell phenotype. These biological mediators of cellular responses are potentially interactive and as such could drive cells through progressive phenotypes to create new tissue as in morphogenesis and wound repair. In fact, ECM composition changes during tissue formation accompanied by alterations in cell growth and migration. How alterations in the ECM regulate cell activities is poorly defined. To address this question in wound repair, we cultured normal human dermal skin fibroblasts in relaxed collagen gels, fibronectin-rich cultures or stressed fibrin gels, and stressed collagen gels to model normal dermis, early wound provisional matrix, and late granulation tissue, respectively. Integrin subunits, alpha 2, alpha 3, and alpha 5, that define receptor specificity for collagen and provisional matrix, respectively, were measured at mRNA steady-state level before and after stimulation with platelet-derived growth factor-BB (PDGF-BB), a potent mitogen and chemoattractant for fibroblasts. Fibronectin-rich cultures and fibrin gels supported PDGF-BB induction of alpha 3 and alpha 5 mRNA. In contrast, both stressed and relaxed collagen attenuated these responses while promoting maximal alpha 2 mRNA expression. Posttranscriptional regulation was an important mechanism in this differential response. Together PDGF-BB and collagen gels promoted alpha 2, but not alpha 3 and alpha 5, mRNA stability. Conversely, when fibroblasts were in fibronectin-rich cultures, PDGF-BB promoted alpha 3 and alpha 5, but not alpha 2, mRNA stability. We suggest that ECM alterations during wound healing or any new tissue formation cause cells to respond differently to repeated growth factor stimuli. An ordered progression of cell phenotypes results, ultimately consummating tissue repair or morphogenesis.

Animals↗
PubMed1997

A three-dimensional collagen lattice induces protein kinase C-zeta activity: role in alpha2 integrin and collagenase mRNA expression.

A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor alpha2 subunit mRNA expression in both unstimulated or platelet-derived growth factor-stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239-249). Here we report that the collagen gel can signal protein kinase C (PKC)-zeta activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-zeta immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-zeta protein levels or intracellular location was observed. DNA binding activity of nuclear factor kappaB (NF-kappaB), a downstream regulatory target of PKC-zeta, was also increased by fibroblasts grown in collagen gel. The composition of the NF-kappaB/Rel complexes that contained p50, was not changed. The potential role of PKC-zeta in collagen gel-induced mRNA expression of collagen receptor alpha2 subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of alpha2 and MMP-1 mRNA in collagen gel-stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)-inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5' end of PKC-zeta mRNA sequences significantly reduced the collagen lattice-stimulated alpha2 and MMP-1 mRNA levels. Taken together, these data indicate that PKC-zeta, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for alpha2 and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-zeta.

Antigens, CD↗
PubMed1999

Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells.

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860-28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I-triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.

Animals↗
PubMed2000

Differential regulation of p27(Kip1) expression by mitogenic and hypertrophic factors: Involvement of transcriptional and posttranscriptional mechanisms.

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G(1) cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G(1) phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27(Kip1) expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27(Kip1) protein. The time course of p27(Kip1) decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27(Kip1) synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-beta1 by aortic SMC. These results identify p27(Kip1) as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.

Angiotensin II↗
PubMed2006

The G534E polymorphism of the gene encoding the factor VII-activating protease is associated with cardiovascular risk due to increased neointima formation.

The G534E polymorphism (Marburg I [MI]) of factor VII-activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Actins↗
PubMed2001

Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Adenoviridae↗
PubMed2000

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Animals↗
PubMed2001

Development of a three-dimensional transmigration assay for testing cell--polymer interactions for tissue engineering applications.

The ability of synthetic or natural scaffolds to support invasion of cells from surrounding tissue is a key parameter for tissue engineering (TE). In this study, the migration of fibroblasts, chondrocytes, and osteoblasts into biodegradable polymer scaffolds was evaluated using a novel, three-dimensional (3-D) transmigration assay. This assay is based on a cell-populated contracted collagen lattice with a biodegradable polymer scaffold implanted at the center of the collagen gel. Cell migration into the scaffolds was assessed both quantitatively and qualitatively following various time lengths in culture using image analysis. Chondrocytes, incorporated within the collagen lattice, migrated into polymer scaffolds, when cultured both statically or in a rotating bioreactor. However, the bioreactor cultures resulted in a significantly greater cell invasion as compared to static cultures. There was a cell density-dependent osteoblast migration from collagen lattice into polymer scaffold, when tested in the transmigration assay. In addition, polymer scaffolds, treated with or without recombinant human platelet-derived growth factor (rh-PDGF-BB) were evaluated for fibroblast migration. The presence of rh-PDGF-BB resulted in significantly greater fibroblast invasion as compared to untreated scaffolds. Our studies suggest that the transmigration model provides a rapid system for testing cell invasion of potential scaffolds for tissue engineering applications.

Becaplermin↗
PubMedDate not supplied

Stimulation of vascular smooth muscle cell migration by macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor that influences the migration and proliferation of various cell types, predominantly monocytes and macrophages. Recent evidence suggests an important role for MIF in the progression of atherosclerosis and restenosis. For this reason, we studied the effect of MIF on platelet-derived growth factor-BB (PDGF-BB)-induced migration and PDGF receptor protein expression in vascular smooth muscle cells (VSMCs). Furthermore, the possibility of MIF influencing the migration of VSMCs was investigated. Our results show that short-term incubation of MIF is able to enhance PDGF-BB-induced migration. Long-term incubation decreases PDGF-BB-induced migration, but preserves a short-term stimulatory effect. These effects are not regulated at the level of PDGF receptor protein expression. MIF also acts as a chemoattractant for VSMCs, with a maximum response at 15 ng/ml. In contrast, the proliferation of VSMCs was unaffected by MIF. We conclude that MIF has a biphasic effect on VSMC migration. It remains unclear whether this effect is direct or involves the secretion of unidentified promigratory factors. Exogenous MIF does not stimulate VSMC proliferation; however, a role for MIF in proliferation cannot be fully ruled out. In view of the known key contributions of macrophage-derived MIF and VSMCs, the observed effects may well play a role in the progression of atherosclerosis and restenosis.

Animals↗
PubMed2006

Effect of rhPDGF-BB delivery on mediators of periodontal wound repair.

Growth factors such as platelet-derived growth factor (PDGF) exert potent effects on wound healing including the regeneration of tooth-supporting structures. This investigation examined the effect of the local delivery of PDGF-BB when combined with reconstructive periodontal surgery on local wound fluid (WF) levels of PDGF-AB, vascular endothelial growth factor (VEGF), and bone collagen telopeptide (ICTP) in humans with advanced periodontitis. Sixteen patients exhibiting localized periodontal osseous defects were randomized to one of three groups (beta-TCP carrier alone, beta-TCP + 0.3 mg/mL of recombinant human PDGF-BB [rhPDGF-BB], or beta-TCP + 1.0 mg/mL of rhPDGF-BB) and monitored for 6 months. WF was harvested and analyzed for PDGF-AB, VEGF, and ICTP WF levels. Teeth contralateral to the target lesions served as controls. Increased levels of VEGF in the WF was observed for all surgical treatment groups with the 1.0 mg/mL rhPDGF-BB group showing the most pronounced difference at 3 weeks in the AUC analysis versus control (p < 0.0001). PDGF-AB WF levels were increased for the carrier alone group compared to both rhPDGFBB groups. Low-dose rhPDGF-BB application elicited increases in ICTP at days 3-5 in the wound healing process, suggesting a promotion of bone turnover at early stages of the repair process (p < 0.02). These results demonstrate contrasting inducible expression patterns of PDGF-AB, VEGF, and ICTP during periodontal wound healing in humans.

Adult↗
PubMed1999

Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

3T3 Cells↗
PubMed2000

Regulation of macropinocytosis by p21-activated kinase-1.

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

3T3 Cells↗
PubMed1998

Cyclin D3-associated kinase activity is regulated by p27kip1 in BALB/c 3T3 cells.

We report that cyclin D3/cdk4 kinase activity is regulated by p27(kip1) in BALB/c 3T3 cells. The association of p27(kip1) was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27(kip1)/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27(kip1)-specific antibodies. The association of p27(kip1) with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27(kip1) protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27(kip1) kinase activity was present throughout the cell cycle. Since we found that the p27(kip1) activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27(kip1) and the active cyclin D3 lacking p27(kip1) by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27(kip1)/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27(kip1) has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.

3T3 Cells↗
PubMed2003

Mechanism of human dermal fibroblast migration driven by type I collagen and platelet-derived growth factor-BB.

Migration of human dermal fibroblasts (HDFs) is critical for skin wound healing. The mechanism remains unclear. We report here that platelet-derived growth factor-BB (PDGF-BB) is the major promotility factor in human serum for HDF motility on type I collagen. PDGF-BB recapitulates the full promotility activity of human serum and anti-PDGF neutralizing antibodies completely block it. Although collagen matrix initiates HDF migration without growth factors, PDGF-BB-stimulated migration depends upon attachment of the cells to a collagen matrix. The PDGF-BB's role is to provide directionality and further enhancement for the collagen-initiated HDF motility. To study the collagen and PDGF-BB "dual signaling" in primary HDF, we establish "gene cassettes" plus lentiviral gene delivery approach, in which groups of genes are studied individually or in combination for their roles in HDF migration. Focal adhesion kinase, p21(Rac,CDC42)-activated kinase and Akt are grouped into an upstream kinase gene cassette, and the four major mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 5) are grouped into a downstream kinase gene cassette. The experiments demonstrate 1) the genes' individual roles and specificities, 2) their combined effects and sufficiency, and 3) the mechanisms of their intermolecular connections in HDF migration driven by collagen and PDGF-BB.

Becaplermin↗
PubMed1994

Myelination in vitro of rodent dorsal root ganglia by glial progenitor cells.

Oligodendrocytes synthesize myelin in the mammalian central nervous system; they develop from glial progenitors which, at least in vitro, are bipotential and also differentiate into astrocytes. Maturation of these O-2A progenitors is known to be influenced by growth factors and by extracellular matrix molecules. We investigated the effect of neurons on glial development by co-culturing highly purified rodent embryonic dorsal root ganglia with neonatal O-2A progenitors. Neurons produce signals, including platelet-derived growth factor BB and basic fibroblast growth factor, which stimulate progenitor cells to synthesize DNA; axonal contact is associated with down-regulation in the expression of complex ganglioside surface molecules on O-2A progenitors; with maturation, many of these cells develop into oligodendrocytes allowing the normal process of myelination to take place, but neurons also promote the differentiation of type 2 astrocytes. This orchestration of proliferation and differentiation in O-2A progenitor cells favours the development of glial-neuronal interactions needed for saltatory conduction of the nerve impulse.

Animals↗