Research Report - Nikolaus-Fiebiger-Zentrum für Molekulare Medizin

Increased TGF-� signaling in the cardiovascular system has also been found in mice
hypomorphic for fibulin-4 and in the patient positive FBLN4 mutation. Recently, we
found fibulin-4 expression also in fetal growth plate cartilage. Preliminary data on
Fbln4 null mice showing reduced bone mass and increased bone fragility, and
mutations found in human patients indicate that fibulin-4 may be also involved in
skeletal development. The goal of this study is to obtain further insight into molecular
changes causing the observed of skeletal alterations in fibulin-4 deficient mice such
as limb contracture, detached distal phalanges and reduced bone mass.
Results:
Biochemical analyses on bone revealed that collagen I from fibulin-4 null mice was
less cross-linked compared with those from wild type and heterozygous mice.
Reduction of proteolytic activation of lysyl oxidase (LOX) which is essential for
collagen crosslinking was detected not only in osteoblast culture but also in
chondrocyte and fibroblast culture from fibulin-4 null mice. The mechanism how
fibulin-4 is involved in LOX activation will be elucidated in vitro. It is still necessary to
quantify the crosslink markers hyroxylyslpyridinoline (HP)/lysylpyridinoline (LP) of
some more samples.
Fig.5 SDS Page of collagens extracted with
acid form bones of fibulin-4 k.o. mice
shows significantly enhanced amounts of
soluble collagen chains (a1(I) and a2(I)) ,
indicating impaired crosslink formation,
while collagens from wild type or
heterozygote bones remain mostly insoluble
2) There is ample evidence in the literature for a role of fibulin-4 in extracellular
assembly of collagen and elastin. Therefore, particular attention was paid to fibulin-4
binding proteins such as LTBPs and lysyloxidases in order to elucidate the
mechanisms by which the absence of fibulin-4 leads to abnormalities in mice and
human. To this aim, four mutations in fibulin-4 found in human patients were
introduced in recombinant fibulin-4. The C276Y mutant fibulin-4 was not secreted
from transfected 293 cells indicating that this cysteine residue is important for the
protein folding. Other mutants can be secreted and purified except R279C mutant.
These data suggest that the homozygous missense mutation of C276Y and the
compound heterozygosity resulted in null mutation of fibulin-4 therefore those
patients died very early, similar to fibulin-4 null mice. It will be tested whether cells
other than 293 cells can secrete mutant fibulin-4. Some of fibulin-4 mutants exhibited
reduced binding activities with different ligands. The E57K and E126K mutants were
more susceptible to the proteases tested, suggesting that these mutation resulted in
loss of calcium, causing instability in the conformation of EGF-like domain .
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