Regulation of proximal tubular epithelial cell CD44-mediated binding and internalisation of hyaluronan.
BACKGROUND
Increased expression of the connective tissue polysaccharide hyaluronan (HA) in the renal corticointerstitium is associated with progressive renal fibrosis. Numerous studies have demonstrated involvement proximal tubular epithelial cells in the fibrotic process and in the current study we have characterised their expression of the HA receptor, CD44, and examined changes in CD44 expression and function in response to either IL-1beta or glucose.


METHODS
Characterisation of CD44 splice variant expression was carried out in primary cultures of human proximal tubular cells (PTC) and HK2 cells. Binding and internalisation HA was examined by addition of exogenous of fluorescein-HA (fl-HA), and expression of CD44 examined by immunoblot analysis and flow cytometry. Alteration in "functional" CD44 was determined by immunoprecipitation of CD44 following stimulation in the presence of fl-HA.


RESULTS
PTC, both primary culture and the PTC cell line, HK2, express at least 5 CD44 splice variants, the expression of which are not altered by addition of either IL-1beta or 25mM D-glucose. Addition of either stimulus increased cell surface binding and internalisation of fl-HA and increased expression of functionally active CD44. Increased binding and internalisation of fl-HA, was blocked by anti-CD44 antibody, and by the inhibition of O-glycosylation.


CONCLUSIONS
The data demonstrate that stimuli inducing PTC HA synthesis also regulate PTC-HA interactions. Furthermore increased HA binding and internalisation is the result of post-translational modification of CD44 by O-glycosylation, rather than by alteration in expression of CD44 at the cell surface, or by alternate use of CD44 splice variants.
Jones
Stuart George
SG
Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, UK.
Ito
Takafumi
T
Phillips
Aled Owain
AO
D001692:Biological Transport; D002478:Cells, Cultured; D004705:Endocytosis; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis*; D006820:Hyaluronic Acid / Q000096:biosynthesis* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000276:immunology / Q000378:metabolism*
D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid