Signal Transduction Pathways Involved in the Mitogenic Activity of Pleiotrophin

Abstract

Pleiotrophin (PTN) is a developmentally regulated protein which exhibits neurite-outgrowth, mitogenic, and angiogenic properties. It has also been shown to be involved in tumor growth and metastasis. Here we used primary BEL (bovine epithelial lens) cells to investigate the signal transduction pathways involved in the mitogenic activity of recombinant PTN. PTN was purified from conditioned media of SW-13 cells transfected with the human PTN cDNA. We show that inhibitors of tyrosine kinase, mitogen-activated protein kinase, or phosphoinositide (PI) 3-kinase inhibit DNA synthesis stimulated by PTN. Analysis of tyrosine-phosphorylated proteins following PTN stimulation showed phosphorylation of two novel 190-and 215-kDa proteins in addition to SHC, ERK1, and ERK2. A mobility shift of phosphorylated ERK1 and ERK2 was detected with a panERK antibody confirming the phosphorylation of the two ERKs. Furthermore, in vitro immunocomplex kinase assay with Akt1, a natural substrate of PI 3-kinase, showed an activation of the kinase following PTN stimulation and a reversal by the PI 3-kinase inhibitor wortmannin. We conclude that the mitogenic activity of PTN is dependent on tyrosine kinase activation and utilizes the mitogen-activated protein kinase and the PI 3-kinase pathways to transduce a mitogenic signal. Pleiotrophin (PTN)1 (1) is an 18-kDa secreted protein belonging to a new family of heparin-binding factors which includes midkine (2) and the chicken retinoic-inducible heparin-binding protein (3). Both, PTN and midkine are developmentally regulated proteins which exibit neurite outgrowth, angiogenic and mitogenic properties (reviewed in Ref. 4), and which enhance plasminogen activator activity (5). PTN and midkine share 50% of sequence homology, but they have no homology with the fibroblast growth factor family of heparin-binding growth factors which display several overlapping biological properties (reviewed in Ref. 6).The mitogenic activity of PTN is a subject of controversy as conflicting results were reported by different laboratories. On one hand, PTN purified from tissues or expressed in bacteria and in insect cells is devoid of mitogenic activity but still promotes neurite outgrowth (7-9); however, Courty et al. (10) reported mitogenic activity after purification of PTN from bovine brain. On the other hand when expressed in eukaryotic cells, PTN is capable of stimulating thymidine incorporation and cell growth in different cell systems including fibroblasts (11), epithelial cells (11,12), and endothelial cells (11, 13). The failure of purifying mitogenically active PTN in the former systems listed above may be explained by either loss of mitogenic activity during the purification or erroneous folding of the protein when highly expressed. This latter explanation would be in agreement with the findings of Laaroubi et al. (13) who demonstrated that PTN expressed in NIH3T3 cells is produced in two forms distinct in their structure and properties. These two forms can be separated in two peaks on a Mono S column;...

Haptotactic Migration Induced by Midkine

“…Consistent with our data, Souttou et al reported that Erk1 and 2 and PI3-kinase are involved in PTN/HB-GAM-mediated cell proliferation (57). In addition, Src, JAK1, and 2 and ␤-catenin have been reported to be involved in PTN/HB-GAM signaling (58,59,60).…”

Pleiotrophin, an angiogenic and mitogenic growth factor, is expressed in human gliomas

“…PTN has been reported to be mitogenic for endothelial and epithelial cells, as well as for fibroblasts (Li et al 1990;Fang et al 1992;Delbe et al 1995;Souttou et al 1997Souttou et al , 2001). However, also antiproliferative effects on chondrocytes have been reported (Tapp et al 1999).…”

“…Mature PTN is a non-glycosylated, lysine-rich peptide that migrates anomalously as an 18-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (therefore also termed p18; Corbley 1997) and is derived from a 168-residue precursor with a 32 amino acid signal sequence. PTN is mitogenic for various cell types, it promotes angiogenesis, stimulates neurite outgrowth from cultured neurones, and induces cell migration (Li et al 1990;Fang et al 1992;Delbe et al 1995;Souttou et al 1997Souttou et al , 2001Rauvala et al 2000). Together with midkine (MK) it forms a family of heparin-binding proteins that are normally expressed during embryogenesis, but rarely in healthy adult tissues (Iwasaki et al 1997).…”

Dominant Negative Effectors of Heparin Affin Regulatory Peptide (HARP) Angiogenic and Transforming Activities

“…The specificity of the H⌬111-136 inhibitory effect was also tested with the structurally related molecule MK, which displayed no effects upon 2 nM HARP stimulation (data not shown). Because the HARP mitogenic signal, at least in BEL and NIH-3T3 cells, is transduced through the MAP kinase signaling pathway leading to the phosphorylation of ERK1 and ERK2 (22,27), the ability of HARP to induce MAP kinase phosphorylation in the presence of H⌬111-136 was evaluated (Fig. 1B).…”