Pergamon Cellular Signalling Vol. 6, No. 2, pp. 173-180, 1994. ~ght © 1994 Elsevier Science L ~ Printed in Great Britain. All fights r(~rv~l 0898-6568/94 $7.00 + 0.00 0898-6568(93)E0002-E DIFFERENT PHOSPHORYLATED FORMS OF INOSITOLPHOSPHATE GLYCAN COULD BE INVOLVED IN THE TRANSFORMING GROWTH FACTOR-~ 1 (TGF-~I1) SIGNALLING PATHWAY DENIS VIVIEN, PATRICK BOGDANOWlCZ, KARIM BOUMEDIENE, LAURENT MARTINY,* BERNARD HAYE,* and JEAN-PIERRE PUJOLt Laboratoire de Biochimie du Tissu Conjonctif, CJF INSERM 91-06, CHU Caen Ctte de Nacre, 14033 Caen, France and *Laboratoire de Biochimie, UFR Sciences, Universit6 de Reims, DRED-EA1243 et CNRS-ERS FOOl7, 51062 REIMS, France (Received 14 July 1993; and accepted 1 October 1993) Abstract--Labelling with [3H]ghcosamine was used to prepare a transforming growth factorq31 (TGF-~l)-sensi- tive glycosylphosphatidylinositol (GPI) from monolayer cultures of rabbit articular chondrocytes (RAC), which may be involved in control of the cell cycle. The polar headgroup of this glycosylphosphatidylinositol was generated by both phosphatidylinositol-specific phospholipase C (PI-PLC) and pronase E digestion. The molecule emerged in only one peak on a Dowex AG1-X8 chromatogram, eluted at 0.1 N ammonium formate. In contrast, similar experi- ments performed on cellular extract from cultures previously labelled with [3H]glucosamine displayed four radio- active peaks eluting at 0.1, 0.2, 0.5 and 1 N ammonium formate, respectively. Evidence that the eluting position of these peaks was dependent on the number of phosphate residues present in each fraction was demonstrated by both [32p]phosphorus labelling and change in the position of alkaline phosphatase-induced shift in elution volume. We also demonstrated that the GPI-derived inositolphosphate glycan (IPG) could be hyperphosphorylated into the cell under the action of a kinase whose activity was enhanced by TGF-I~I itself. We have also shown that all of these IPG forms could mimic the TGF-13-inducedincrease of DNA replication rate of RAC, with a higher activity for peaks HI and IV than peaks I and H. Key words: Transforming growth factor-131 mediators, articular chondrocytes, inositolphosphate glycan, kinase activity. INTRODUCTION RECErer results have shown that an insulin-sensi- tive glycosylphosphatidylinositol (GPI) could mimic several actions of this factor such as: lipol- ysis [1], lipogenesis [2], effects on pyruvate kinase and cyclic AMP [3]. Other similar events have been reported for the biological action of interleukin 2 [4], nerve growth factor [5] and thyrotropin [6]. We have also shown that a GPI- tAuthor to whomcorrespondence shouldbe addressed. Abbreviations: TGF--transforming growth factor; GPI glycosylphosphatidylinositol; RAC---rabbitarticular chondro- cytes; PI-PLC--phosphatidylinositol specific phospholipase C; IPG--inositolphosphate glycan; DMEM--Dulbecco's ModifiedEagle's Medium;FCS--foetalcalf serum. anchored molecule could be involved in the mech- anism of action of transforming growth factor (TGF-[)I) on the cell cycle control of rabbit artic- ular chondrocytes (RAC) [7,8]. TGF-[~I was shown to stimulate the release of inositolphos- phate glycan (IPG) from a corresponding lipid precursor. Moreover, the polar headgroup mimics the TGF-[S1 effect on RAC growth promotion. We have also demonstrated in a previous study, that a membrane hydrophobic phase obtained by Triton X-114 partitioning contained a GPI-anchored molecule and that this extract was sensitive to both phosphatidylinositol-specific phospholipase C (PI-PLC) and TGF-[31 treatment [8]. Despite the current understanding of the com- position of IPG mediators, their precise structures 173