Production of heparan sulphate proteoglycans by human bone marrow stromal cells.
Haemopoietic progenitors from human bone marrow bind strongly to human marrow stromal cell cultures but the interaction only occurs if the stromal cells are maintained in methyl prednisolone. Heparan sulphate has been implicated in this interaction and in the binding of haemopoietic cell growth factors. In the present study we have compared the molecular structures of the heparan sulphate proteoglycans, metabolically labelled with [35S]sulphate, produced by methyl prednisolone-treated and untreated human marrow stromal cells in vitro. [35S]proteoglycans were examined in the cell layers (extracted with 1% (v/v) Triton X-100 in 6 M urea) and in the culture medium. Fractionation of proteoglycans by ion-exchange chromatography indicated that the heparan sulphate produced by the treated cultures eluted at a higher NaCl concentration than the counterpart from untreated cells. The heparan sulphate appeared to be mainly expressed on the cell surface, since it was efficiently extracted by treatment with dilute trypsin (50 micrograms ml-1 for 10 min). All cultures contained two heparan sulphate proteoglycan species, the major component eluted from a Sepharose CL-4B column with a median Kav of 0.33 and apparently contained an average of only one heparan sulphate chain. Small quantities of a larger proteoglycan, which was eluted in the void volume from the CL-4B column, was also detected, mainly in the cell layer extracts. The molecular structure of the heparan sulphate chains was analysed by oligosaccharide mapping, following specific enzymic depolymerisation, and separation of breakdown products by gradient PAGE. The maps revealed significant differences in overall enzyme susceptibilities and sulphation patterns of polysaccharides produced by methyl prednisolone-treated and untreated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Morris
A J
AJ
Cancer Research Campaign Department of Medical Oncology, Christie Hospital & Holt Radium Institute, Manchester, UK.
Turnbull
J E
JE
Riley
G P
GP
Gordon
M Y
MY
Gallagher
J T
JT
D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D006025:Glycosaminoglycans / Q000032:analysis; D006410:Hematopoiesis / Q000502:physiology*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D006801:Humans; D009844:Oligosaccharides; D011509:Proteoglycans / Q000032:analysis / Q000096:biosynthesis*; D014357:Trypsin
D003470:Culture Media; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D009844:Oligosaccharides; D011509:Proteoglycans; D014357:Trypsin