ABSTRACT
HB-GAM/Pleiotrophin and Midkine (MK) are developmentally-regulated proteins with putative functions during cell growth and differentiation. Using the P19 cell which is a model to study the events associated with early development, we examined the expression and cellular localization of HB-GAM and MK during neural differentiation of P19 cells induced by retinoic acid (RA). The temporal expressions of HB-GAM and MK transcripts and both the levels and cellular localizations of the corresponding proteins appeared dramatically different. MK mRNA, already expressed in untreated P19 cells, was transiently increased by exposure to RA and then largely down regulated. More interestingly, HB-GAM which was not detected in untreated P19 cells was strongly expressed after 2 days of RA treatment and this expression persists throughout the duration of the culture suggesting that it could be involved in different aspects of this differentiation process.
Subject(s)
Carrier Proteins/biosynthesis , Cytokines/biosynthesis , Neurons/cytology , Tretinoin/pharmacology , Animals , Blotting, Northern , Carcinoma, Embryonal/metabolism , Cell Differentiation , Cell Line , Cell Line, Tumor , Choline O-Acetyltransferase/metabolism , DNA/chemistry , DNA/metabolism , DNA, Complementary/metabolism , Extracellular Matrix/metabolism , Humans , Immunohistochemistry , Keratolytic Agents/pharmacology , Membrane Glycoproteins/biosynthesis , Mice , Midkine , Neurons/metabolism , Proteoglycans/biosynthesis , RNA, Messenger/metabolism , Recombinant Proteins/chemistry , Syndecan-3 , Time Factors , Tretinoin/metabolismABSTRACT
Heparin affin regulatory peptide (HARP) is an heparin-binding growth factor, highly expressed in several primary human tumors and considered as a rate-limiting angiogenic factor in tumor growth, invasion, and metastasis. Implication of this protein in carcinogenesis is linked to its mitogenic, angiogenic, and transforming activities. Recently, we have demonstrated that the C-terminal residues 111-136 of HARP are required for its mitogenic and transforming activities (Bernard-Pierrot, I., Delbe, J., Caruelle, D., Barritault, D., Courty, J., and Milhiet, P. E. (2001) J. Biol. Chem. 276, 12228-12234). In this paper, HARP deleted of its last 26 amino acids was shown to act as a dominant negative effector for its mitogenic, angiogenic, transforming, and tumor-formation activities by heterodimerizing with the wild type protein. Similarly, the synthetic corresponding peptide P111-136 displayed in vitro inhibition of wild type HARP activities, but in this case, the inhibition was mainly explained by the competition of the peptide with HARP for the binding to the extracellular domain of the high affinity ALK receptor.
Subject(s)
Carrier Proteins/physiology , Cell Transformation, Neoplastic , Cytokines/physiology , Endothelium, Vascular/physiology , Growth Substances/physiology , Neoplasms/prevention & control , Neovascularization, Physiologic/physiology , Peptide Fragments/pharmacology , 3T3 Cells , Amino Acid Sequence , Animals , Antineoplastic Agents/pharmacology , Aorta , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cytokines/chemistry , Cytokines/genetics , DNA Replication , Endothelium, Vascular/drug effects , Humans , Kinetics , Mice , Mice, Nude , Molecular Sequence Data , Neoplasms/blood supply , Neovascularization, Physiologic/drug effects , Peptide Fragments/chemistry , Recombinant Proteins/metabolismABSTRACT
In the kidney, in which development depends on epithelial-mesenchymal interactions, it has been shown that retinoids modulate nephrogenesis in a dose-dependent manner in vivo and in vitro. Midkine (MK) is a retinoic acid responsive gene for a heparin-binding growth factor. The aim of the present study was therefore to quantify the expression of MK mRNA during renal development in the rat, to analyze the regulation of MK expression by retinoids in vivo and in vitro, and, finally, to study the role of MK in rat metanephric organ cultures. The spatiotemporal expression of MK in fetal kidney was studied. In control rats, MK expression is ubiquitous at gestational day 14, i.e., at the onset of nephrogenesis. On day 16, MK is expressed in the condensed mesenchyme and in early epithelialized mesenchymal derivatives. On gestational day 21, MK is rather localized in the nonmature glomeruli of the renal cortex. In utero exposure to vitamin A deficiency did not modify the specific spatial and temporal expression pattern of MK gene in the metanephros, although a decrease in mRNA expression occurred. In metanephroi explanted from 14-d-old fetuses and cultured in a defined medium, expression of MK mRNA was found to be stimulated when retinoic acid (100 nM) was added in the culture medium. Finally, in vitro nephrogenesis was strongly inhibited in the presence of neutralizing antibodies for MK: the number of nephrons formed in vitro was reduced by approximately 50% without changes in ureteric bud branching morphogenesis. These results indicated that MK is implicated in the regulation of kidney development by retinoids. These results also suggested that MK plays an important role in the molecular cascade of the epithelial conversion of the metanephric blastema.