Gc-globulin (vitamin D binding protein) is synthesized and secreted by hepatocytes and internalized by hepatic stellate cells through Ca(2+)-dependent interaction with the megalin/gp330 receptor.
BACKGROUND
Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture.


METHODS
Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [(35)S]-methionine, and its intracellular binding to alpha-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and alpha-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis.


RESULTS
Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [(35)S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca(2+), suggesting a Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and alpha-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to alpha-smooth-muscle actin filaments was confirmed by co-immunoprecipitation.


CONCLUSIONS
We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to alpha-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.
Gressner
Olav A
OA
Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Aachen, Germany. ogressner@ukaachen.de
Lahme
Birgit
B
Gressner
Axel M
AM
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D017931:DNA Primers; D022781:Hepatocytes / Q000378:metabolism*; D047468:Immunoprecipitation; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D014809:Vitamin D-Binding Protein / Q000096:biosynthesis* / Q000378:metabolism
D017931:DNA Primers; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D014809:Vitamin D-Binding Protein; D002118:Calcium