Measuring Interactions Between ECM and TGFB-Like Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 222

region, rich in proline and basic amino acids, suggesting that proteolytic cleav-
age may be the mechanism for release of LTBP1 from matrix. A number of in
vitro studies have confirmed that LTBP1 (together with its bound latent TGF )

can be released from matrix by various proteases (9-12) . However, these stud-
ies have focused on examining release by addition of purified exogenous pro-
teases. To date, no technique has been described for measuring release of
LTBP1/latent TGF

β

by cells during the process of matrix remodeling. Because
the release of growth factors from matrix-bound stores may be a key step in
regulating their activity, a method for examining this would be of great benefit
to researchers in the field. The following account details an immunoprecipitation
method for examining the release of matrix-bound LTBP1/latent TGF

β

by cells
during matrix remodeling. The example described examines the release of
LTBP1/latent TGF

β

by osteoclasts from matrix laid down by osteoblasts. How-
ever, the assay could readily be adapted to examine release of LTBP1/latent
TGF

β

by any one cell type from the ECM of any other cell type. Furthermore,
as long as a good source of antibodies for immunoprecipitation is available, the
assay could be modified to examine the cell-mediated release of other matrix-
bound growth factors or matrix proteins from ECM.
The principle of the assay is summarized in Fig. 2 . Primary osteoblast cells
are grown in long-term culture to a stage when they are producing an extensive
layer of ECM (10-14 d). The cells are then metabolically labeled for 48 h
using 35 S-cysteine. This labels intracellular and secreted proteins, but also the
ECM proteins. The cells are then lysed, leaving the insoluble matrix adhered to
the culture plate. This radiolabeled matrix is extensively washed and dried.
Avian osteoclast cells are then seeded onto the radiolabeled matrix. Release of
LTBP1 and latent TGF

β

by the osteoclasts during matrix remodeling is moni-
tored by immunoprecipitating the labeled LTBP1 from the culture media (the
LTBP1 antibodies will precipitate both free LTBP1 and LTBP1 complexed to
latent TGF

β

). Because the osteoclast cells were never exposed to 35 S-cysteine,
it is assumed that any radiolabeled proteins present in the culture media must
have been released from the matrix. Any labeled LTBP1 remaining bound in
the matrix (i.e., not released by the cells) can also be measured by performing
a plasmin digestion of the matrix to release LTBP1, followed by immunopre-
cipitation using anti-LTBP1. In this way, it should be possible to correlate
release of LTBP1 and latent TGF

β

by the osteoclasts into the culture media
with a concomitant reduction in LTBP1 and latent TGF

β

β remaining in the

matrix (for example, see Fig. 3 ).

2. Materials
Note: All chemicals used should be at least analytical grade. Where a spe-
cific manufacturer is used for a particular reagent, it is mentioned in the text.