Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth

A rat hepatocyte cell line was cultured in Higuchi's medium with fetal calf serum and insulin and labeled with ³⁵SO₄^2-. The cells were treated with a number of ligands to displace the heparan ³⁵SO₄ proteoglycan (HSPG) from the pericellular matrix. Maximum release was obtained with D-mannose-6-PO₄ (50 mM), D-glucose-6-PO₄ (50 mM), myo-inositol-2-PO₄ (2-5 mM), myo-inositol hexaphosphate (2-5 mM), and DL-myo-inositol-1-PO₄ (1-2 mM). D-myo-Inositol-1,3,4-(PO₄)₃ (1 mM) and L-myo-inositol-1-PO₄ (2 mM) were intermediate in their ability to release the cell surface HSPG, whereas heparin (2 mg/ml), yeast phosphomanan (4 mg/ml), D-xylose-1-PO₄ (50 mM), D-glucose-6-SO₄ (50 mM), and myo-inositol hexasulfate (5 mM) were ineffective. When ³⁵SO₄^2- was added to cell cultures, the total cell surface HSPG increased linearly, but the percentage of the total cell surface [³⁵SO₄] HSPG that was released by myo-inositol-PO₄ increased with time during the labeling period, reaching a maximum of 65% after 5 h. When cells were labeled for 12 h without insulin in the medium, the maximum amount of cell surface HSPG that was released by myo-inositol-PO₄ was reduced to 30%. However, when cells labeled in the absence of insulin were treated with phosphatidylinositol-specific phospholipase C and then myo-inositol-PO₄, the release of the cell surface [³⁵SO₄]HSPG was increased to 73%. When the [³⁵SO₄]HSPG that was released from the cell surface by treatment with myo-inositol-PO₄ was added to cultures of unlabeled hepatocytes, it was taken up very rapidly and a portion of the internalized HSPG was converted to free heparan SO₄ chains which appeared in the nucleus. Uptake was Ca²⁺- and Mg²⁺-independent. The amount of [³⁵SO₄]HSPG taken up was markedly reduced when the myo-inositol-PO₄-releasable [³⁵SO₄]HSPG was pretreated with trypsin, thermolysin, alkaline borohydride, or alkaline phosphatase. When the cells were grown in inositol-deficient medium or in the presence of myo-inositol-PO₄, the amount of heparan SO₄ found in the nucleus was markedly reduced, and the cells no longer exhibited contact inhibition. These effects of myo-inositol deficiency on the growth and nuclear heparan SO₄ were accentuated by addition of LiCl to the cultures to prevent phosphatidylinositol synthesis from the endogenous myo-inositol-PO₄. The results suggest a model for HSPG metabolism in which (a) newly secreted HSPG is initially bound to the plasma membrane through a phosphatidylinositol moiety that is covalently linked to the core protein, (b) the linkage between the core protein and the phosphatidylinositol is then cleaved on the cell surface by an insulin-activated phospholipase C, and (c) the released HSPG which has the myo-inositol-PO₄ moiety still attached to the core protein is then internalized via a cell surface receptor that recognizes the myo-inositol-PO₄ moiety. The results also show that culturing of the hepatocytes under conditions that reduce the amount of nuclear heparan SO₄ results in a loss of contact inhibition.