MIA (Melanoma Inhibitory Activity) was cloned as a
secreted protein from human melanoma cell lines . MIA
is expressed in and secreted from malignant melanomas
and chondrocytes [2, 3]. As MIA expression in chondrocytes
is dependent on the differentiation status of the cells
it was also designated CD-RAP (cartilage-derived retinoic
acid sensitive protein). Retinoic acid is known as a regulator
of chondrocytic cell differentiation in vitro.
Recent results indicate an important role in tumor progression
and metastasis as MIA mediates detachment
of melanoma cells from extracellular matrix molecules
such as fibronectin . MIA expression levels closely parallel the capability of melanoma cells to form metastases
in syngeneic animals. Recently, MIA-deficient mice were shown to have structural
abnormalities in cartilage .
Materials and Methods
Human primary chondrocytes derived from articular cartilage
were cultivated in DMEM supplemented with penicillin
(100 U/ml), streptomycin (10 g/ml) (both Sigma),
and 10 % fetal calf serum (Gibco) under a humidified
atmosphere of 5% CO2 at 37 C, then split 1:2 at 80 % confluency.
As chondrocytes spontaneously dedifferentiate in
cell culture, redifferentiation of the cells was induced by
treatment with transforming growth factor-b (TGF-b) for 4
days. Differentiation was controlled by determining collagen
type II with RT-PCR. Human mesenchymal stem cells
were obtained from BioWhittaker Europe (Poietics) and
cultured as described by the manufacturer.
MIA was measured by a commercially available ELISA
(Roche Applied Science), the manufacturers instructions
were precisely followed and the internal standard
reagents provided were used.
Results and Application
MIA as a potential marker for chondrocyte
differentiation and chondrocytic disorders
High levels of MIA expression were observed in differentiated
chondrocytes. As a result, MIA was also investigated as a potential marker for cartilage tissue engineering
and cartilage damage.
In cell culture of chondrocytes and tissue engineering
of cartilage cell differentiation must be carefully monitored.
The detection of collagen type II as a marker for
chondrocytic differentiation is commonly used.
However, using this marker does create problems as
ELISAs are not feasible, and for RNA analysis, the cells
are lost during extraction procedures. Therefore we
established MIA as a chondrocytic marker.
MIA is a secreted molecule, making it easy to measure its
levels in the cell culture supernatant. Detection by the
MIA-ELISA is highly specific, sensitive, and quantitative.
In cell-culture systems using human chondrocytes
(Figure 1) and human mesenchymal stem cells (Figure 2),
we were able to monitor the process of cell differentiation.
MIA serum concentrations in individuals with different
rheumatic diseases were measured, correlated with
inflammatory parameters and/or with the degree of joint
destruction, and compared with healthy individuals and
subjects suffering from melanoma . Increased MIA
serum concentrations were only found in individuals
with rheumatic diseases associated with joint destruction,
such as rheumatoid arthritis, osteoarthritis, HLA
B27-associated oligoarthritis, and psoriatic arthritis.
Within these rheum
atic diseases, the most significant
increase in MIA serum concentrations was measured in
subjects with rheumatoid arthritis, associated with
rheumatoid factor positivity and joint destruction. Therefore, MIA may be useful to discriminate rheumatoid
arthritis from nondestructive rheumatic diseases.
Further studies are necessary to confirm these preliminary
Our studies indicate that MIA may be a useful marker
for cartilage diseases and the monitoring of chondrocytic
differentiation in cell culture. We conclude that
MIA has the potential to replace other markers due to its
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