R. Zohar et al., SINGLE-CELL ANALYSIS OF INTRACELLULAR OSTEOPONTIN IN OSTEOGENIC CULTURES OF FETAL-RAT CALVARIAL CELLS, Journal of cellular physiology, 170(1), 1997, pp. 88-100
Osteopontin (OPN), a major component of the bone matrix, is expressed
at different stages of bone formation. To determine possible relations
hips between OPN expression and stages of osteogenic cell differentiat
ion, we have performed single cell analyses of intracellular OPN in ea
rly (proliferating), subconfluent (differentiating), and mature (miner
alizing) cultures of fetal rat calvarial cells (FRCC) using a combinat
ion of flow cytometry and confocal microscopy. At each culture stage,
a high proportion (60-98%) of cells were immunoreactive for OPN (OPN+v
e). Each of these populations also included a small proportion of OPN-
ve cells which were characterized by their small size, low granularity
, high proliferative capacity, and enhanced osteogenic potential. The
OPN+ve cells displayed two distinct patterns of intracellular immunost
aining: a perinuclear distribution typical of secreted proteins and a
perimembrane distribution in which patches of OPN were concentrated at
the cell surface. Perimembranous staining predominated in migrant cel
ls, which contained greater than tenfold higher levels of OPN than non
migrant cells as separated in a Boyden chamber. When cell proliferatio
n was high (day 2), most cells were OPN+ve. At all culture stages the
intensity of OPN staining was increased as cells progressed through th
e cell cycle. As cells differentiated and started to form matrix (days
4 and 6), the mean cell expression of OPN was also increased (fourfol
d), independent of changes in total cell protein. However, despite the
association of OPN with osteogenic cells, we were surprised to find t
hat a high proportion (60%) of fetal skin fibroblasts were also immuno
reactive for OPN. The expression of OPN by these cell populations was
confirmed by RT-PCR, and a strong correlation was observed between the
quantitative flow cytometry data and Western blot analysis of cell ex
tracts in which the high and low phosphorylated isoforms of OPN were o
bserved. These studies, therefore, have identified several phenotypes
in FRCC cultures that are based on OPN expression: small OPN-ve cell p
opulations enriched in osteogenic precursors, differentiating osteogen
ic cells that synthesize and secrete OPN, and migrating stromal cells
characterized by a perimembranous OPN staining pattern. (C) 1997 Wiley
-Liss, Inc.