A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain.

313

213

Abstract. Rat sciatic nerve Schwann cells in culture respond to a limited range of mitogens, including glial growth factor, transforming growth factors beta-1 and beta-2 (TGF-fll, TGF-/~2), some cell membrane-associated factors, and to agents such as cholera toxin and forskolin which raise intracellular levels of cAME These responses require the presence of FCS, which exhibits little or no mitogenic activity in the absence of other factors. However, we recently found that forskolin greatly potentiates the mitogenic signal from TGFs-/~I and/32, raising the possibility that cAMP might couple other factors to mitogenesis. We have therefore screened a range of candidate mitogens using DNA synthesis assays. Other than TGFs-fl and glial growth factor, none of the factors tested were mitogenic in the presence of 10% serum alone. With the addition of forskolin, however, porcine PDGF, human PDGF, acidic and basic FGF were potent mitogens for rat Schwann cells, stimulating DNA synthesis and increasing cell number. Cholera toxin and dibutyrylcyclicAMP, but not 1,9-dideoxyforskolin, can substitute for forskolin indicating that the mitogenic effect is mediated via adenylyl cyclase activation. Porcine PDGF gave half-maximal stimulation at 15 pM, and human PGDF an equivalent response at 1 nM. Basic FGF was half maximal at 5 pM, acidic FGF at 1 nM.The recognition of PDGFs and FGFs as mitogens for Schwann cells has many implications for the study of Schwann cell proliferation in the development and regeneration of nerves, and in Schwann cell tumorigenesis.S CHWANN cells, the glial cells of the peripheral nervous system, surround all peripheral nerve axons, and in the case of myelinated nerve fibers, elaborate the myelin sheaths necessary for fast impulse conduction. There are many important developmental and functional interactions between Schwann cells and axons. Of particular interest for this paper is the proliferation of Schwann cells in three contexts. During development, Schwann cells proliferate and migrate out along newly formed axons, surrounding them, and myelinating them in some cases (3,32). If the nerve is injured, through mechanical trauma, neurotoxins or demyelinating diseases for example, Schwann cells again proliferate to restore the integrity of the Schwann cell sheath and aid regeneration of functional nerve fibers (1, 42). Thirdly, Schwann cell tumors may arise on any peripheral nerve, as in the cases of Von Recklinghausen's neurofibromatosis, bilaterai acoustic neurofibromatosis, and spontaneous, nonhereditary acoustic neuromas (47). The origin and nature of the factors involved in these proliferative processes are poorly understood.Attempts to define the factors involved in Schwann cell proliferation have revealed positive responses by various types of Schwann cell in culture to a limited range of mitogens (38, 51), including a few polypeptide growth factors (35), molecules derived from central and peripheral nervous system membranes (13,28,37,39,43,44,49,53), and analogues of cAMP and agents, such as chole...

Section: Forskolin Couples Pdg Fs and Fgfs To Mitogenesismentioning

confidence: 99%

Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells.

Davis

Stroobant

1990

313

213

“…Recombinant PDGF-BB was purified from a Saccharomyces cerevisiae expression system (31). PDGF-AB was purified by immobilized metal ion affinity chromatography from PDGF isolated from human platelets (16). Alternatively, CHO cells expressing recombinant PDGF-AB were used as a source for purification (32).…”

Section: ~L-pdgf-binding Experimentsmentioning

confidence: 99%

Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor.

Sorkin

Westermark

Heldin

et al. 1991

Self Cite

158

Abstract. The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF/3-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the fl-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype fl-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex. A~ajor part of the mitogenic activity in human serum for mesenchymally derived cells, is contributed by PDGF (for reviews see references 20, 36). PDGF consists of disulphide-bonded dimers of homologous A and B polypeptide chains, which combine to create the three possible isoforms PDGF-AA, -AB, and -BB (1, 25, 26). Two PDGF receptor types, which interact with the three PDGF isoforms with different affinities, have been identified (18,19). The PDGF fl-receptor binds PDGF-BB with high affinity and PDGF-AB with lower affinity, whereas the PDGF o~-receptor binds all three ligands with high affinity. cDNAs for both the or-receptor and the/~-receptor have been isolated (7,9,15,28,43). The receptors belong to the family of growth factor receptors equipped with a ligand-stimulatable tyrosine kinase activity, and they are structurally very similar. Their extracellular parts contain five immunoglobulin-like domains and their tyrosine kinase domains are split into two segments by the insertion of ~100 amino acids. Binding of ligand has been shown to induce receptor dimerization, creating homo-as well as heterodimers of PDGF receptors, most likely through the bivalent interaction of one ligand with two receptor molecules (2,17,21,38).Binding of PDGF leads to a rapid internalization of both ligand and receptor (20,36). Studies based on morphological examinations have indicated that at least the ligand is transferred to the lysosomes and degraded after internalizaAlexander Sorkin's permanent address is the Institute of Cytology, Academy of Sciences of USSR, Leningrad 194064, USSR. tion (30,35). However, in other studies, no inhibition of PDGF receptor degradation was recorded after treatment of ceils with lysosomotropic drugs (23,27). Studies of the EGF receptor have shown that it is rapidly inte...

“…The cultures were rinsed three times with cold binding rinse, and bound ~2SI-labeled PDGF was solubilized with 2% Triton X-100. Purified human platelet PDGE consisting primarily ofAB heterodimers (11), was used as the standard. This assay detects PDGF-AA homodimers as well as PDGF-AB heterodimers.…”

Section: Pdgf Radioreceptor Assaymentioning

confidence: 99%

Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-beta.

Majack

Majesky

Goodman

1990

208

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.