ISOLATION AND IDENTIFICATION OF MITOCHONDRIA IN NORMAL HUMAN ARTICULAR CHONDROCYTES
*Noskina, Y; +*Cole, A
+*Rush University at Rush Presbyterian St. Lukes Medical Center, Chicago, IL. Dept. of Biochem., 1653 W. Congress prky, Chicago, IL 60612, 312-942-4879, Fax:
312-942-3053, [email protected]
The focus of this study is to identify the presence and activity of mitochondria and
the presence of apoptotic proteins Bcl-2 and Bax localized to the mitochondrial
membrane in normal adult human chondrocytes.
Introduction: Over the past 30 years glycolysis has been established as the
primary source of energy production in chondrocytes, while mitochondrial energy
production has been thought to have a more minor role in chondrocyte
metabolism. This has been mostly attributed to the low levels of oxygen in the
poorly vascularized cartilage. However most of the work done has been in growth
plate chondrocytes of chickens and rabbits. Work done with articular chondrocytes
has focused on cell metabolism primarily involving matrix synthesis. And cellular
proliferation has been determined by evaluating the levels of DNA synthesis.
Previous studies (1,2) have shown however that at physiologic oxygen tensions
articular chondrocytes displayed a combination of both anaerobic glycolysis and
aerobic mitochondrial metabolism. This study focused on identifying and isolating
mitochondria in normal human articular chondrocytes, which were isolated from
ankle cartilage. Experimental data suggests high levels of mitochondria present in
normal human articular chondrocytes. Besides examining the mitochondria, the
anti-apoptotic protein Bcl-2 localized to the mitochondrial membrane, and Bax a
pro-apoptotic protein of the Bcl-2 family were identified and used as further
markers of the presence of mitochondria. Previous studies on chondrocytes
determined that Bcl-2, was not only present but also appeared be downregulated
under apoptotic conditions in osteoarthritic cartilage (3). It has already been
established that Bcl-2 has a protective effect on mitochondria, and that Bax
localizes to the mitochondria under apoptotic conditions (4). The identification
and isolation of mitochondria and members of the Bcl-2 family of proteins can
lead to further analysis on the involvement of mitochondria in chondrocyte
metabolism under both normal and adverse conditions.
Methods: Human chondrocytes were obtained from normal adult articular ankle
cartilage provided by the Regional Organ Bank of Illinois, within 24 hours of the
death of donors ranging in age from 50 to 70 years old. The cartilage were
digested in 0.2% pronase in Dulbecco's Modified Eagle's Medium (DMEM)
containing 5.0% FBS for 1.5 hours followed by digestion in 0.025% collagenase
in DMEM containing 5.0% FBS for 18 hours. The cells were then rinsed in Hank's
balanced salt solution (HBSS) and pelleted at 1000 g for 10 minutes. Isolated
chondrocytes were also cultured in high-density monolayer in medium containing
10% fetal bovine serum, 45% DMEM, and 45% Ham's F-12, overnight on culture
slides and exposed to the mitochondrial probes MitoTracker  Green and JC-1, to
test for the presence of mitochondria and their activity. The probes were visualized
using fluorescein and rhodamine filters and a fluorometer. The differences the
rhodamine and fluorescein labeled mitochondria were quantitatively confirmed
using the RF-1501 spectrofluorophotormeter (Shimadzu). The isolated
chondrocytes were also fractionated to obtain a nuclear pellet, a crude
mitochondrial pellet, and the soluble cytoplasmic proteins and membrane proteins
supernatant; this was done via the following method. Chondrocytes were
resuspended in a buffer containing 2.5 mM Tris-HCl, pH 7.5, 134 mM NaCl, 5
mM KCl and 0.7 mM Na2HPO4 and collected by centrifugation (5 min at 1000 g
at 4oC). Cells were then swollen for 10 min at 4oC in a hypotonic buffer
containing 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, and 1.5 mM MgCl2, and were
homogenized with 15 strokes in a tight homogenizer. Then 0.4 volume of a cold
2.5 X buffer containing 525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCl,
pH 7.5 and 12.5 mM MgCl2 was added to the cells and they were further
homogenized. This homogenate was centrifuged twice at 1000 g for 10 min at 4oC
to obtain a nuclear pellet (P1). The postnuclear supernatant was centrifuged at
10,000 g for 20 min at 4oC, yielding the crude mitochondrial pellet (P10). The
remaining supernatant contained the cytoplasmic fraction and cellular membranes.
This technique was adapted from that described in Methods in Enzymology (5) for
preparing highly purified mitochondria from Cos-1 African green monkey kidney
cells. Western Blots were performed using nuclear, mitochondrial and cytoplasmic
fractions of the isolated chondrocytes. The fractions were run on 8 % SDS-PAGE
gels under denaturing conditions. The antibodies for western blotting were against
0416
human Bcl-2 (BioSource ) and human Bax, (GibcoBRL ). Antibody binding
was visualized via enhanced chemi-luminescence kit (Amersham ).
Results: Isolated chondrocytes cultured at high-density monolayer and exposed
to the mitochondrial probes depicted high levels of mitochondria as was seen by
the MitoTracker  Green and JC-1 probes. The JC-1 probe used to examine the
mitochondrial membrane potential, depicted a large population of the
mitochondria in chondrocytes to have a high membrane potential. The higher
membrane potential of the mitochondria, seen with a microscope was quantitated
with the use of a fluorometer. The fractionated chondrocyte pellets and the
cytoplasmic supernatant when probed with MitoTracker Green showed the
presence of mitochondria, at the appropriate P10 crude mitochondrial fraction,
thus confirming the successful fractionation of the crude mitochondrial pellet.
Western blotting on the fractions showed the presence of Bcl-2 in the crude
mitochondrial and nuclear fractions while Bax appears to be present in both the
crude mitochondrial fraction and the cytoplasmic proteins supernatant. Further
fractionation of the supernatant to isolate a purified mitochondrial pellet has not
yet been attempted because of the limited number of cells in adult articular
cartilage. This study is designed to ascertain the mitochondrial presence and
activity in normal human articular chondrocytes compared to increasingly
damaged human articular chondrocytes. Therefore the samples of human
articular cartilage are not pooled together but are studied on an individual basis,
making it difficult to obtain enough tissue to perform the further purification
accurately.
Discussion: The presence of high levels of mitochondria in normal human
articular chondrocytes suggests the importance of aerobic metabolism in these
cells even though the availability of oxygen in articular cartilage is decreased
compared to other tissues. It has been previously shown that the anti-apoptotic
protein Bcl-2 is not only expressed in these cells but is also downregulated due to
apoptotic stimuli in osteoarthritic cartilage. Since this protein resides on the
mitochondrial membrane and has been implicated in the protection of these
organelles, it is possible that the mitochondria decrease their activity due to the
lack of Bcl-2 and consequently become apoptotic. Thus while it has been shown
that anaerobic metabolism via glycolysis is the more prevalent form,
mitochondrial metabolism may be necessary for repair. Therefore a decrease in
mitochondrial activity due to adverse conditions, along with possible inhibitors
of the mitochondrial activity may have deleterious effects on articular cartilage.
We have shown here that mitochondria can be isolated from normal articular
human chondrocytes, and that not only is the expression of the anti-apoptotic
protein Bcl-2 present but also the protein itself was visualized via western
blotting. Along with Bcl-2 we have shown the presence of Bax in these cells.
Recent work has shown that Bax while normally present in the cytoplasm of
cells, localizes to the mitochondrial membrane at the onset of apoptosis. The
presence of this protein in the mitochondrial pellet in chondrocytes, may act as a
counter balance to the Bcl-2 on the mitochondria, and thus not only help keep
mitochondrial activity to a minimum, but also keep the cellular activity to a
minimum as well. Future studies will focus on chondrocyte viability in the
presence of not only mitochondrial blockers but also with the increased presence
of both Bcl-2 and Bax.
Acknowledgements: This work is supported in part by the NIH Training Grant
ST32AR07590-4. We also wish to thank Dr. A. Valdellon and his staff at the
Regional Organ Bank of Illinois for the access to human donor cartilage.
References:
1. Lane, et al., Jour Rheum 1977; 4 (4): 334-342.
2. Rajpurohit, et al., Jour Cell Phys 1996; 168: 424-432.
3. Feng, et al., Jour Cell Biochem 1998; 71: 302-309.
4. Woter, et al., Jour Cell Bio 1997; 139: 1281-1292.
5. Casey and Buss, edt. Meth in Enzym (Academic Press) 1995; 250: 464465.
Poster Session - Cartilage Cell Biology - Hall E
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California