Among the most overt negative changes experienced by man and experimental animals under conditions of weightlessness are the loss of skeletal mass and attendant hypercalciuria. These clearly result from some disruption in the balance between bone formation and bone resorption (i.e. remodelling) which appears to be due to a decrease in the functions of the osteoblast. In the studies funded by this project, the clonal osteoblastic cell line, UMR 106-01, has been used to investigate the regulation of collagenase and Tissue Inhibitors of MetalloProteases (TIMPs). This project has shed light on the comprehensive role of the osteoblast in the remodelling process, and, in so doing, provided some insight into how the process might be disrupted under conditions of microgravity

Partridge, Nicola C.

English

NASA Technical Reports Server

NASA-CR-205066
Skeletal Collaqen Turnover by the Osteoblast
/ -/_-'/7-.
Final Technical Report for NAG2-454
Dr. Nicola C. Partridge
St. Louis University School of Medicine
1402 S. Grand
St. Louis, MO 63104
I. Description of Research
Among the most overt negative changes experienced by man and
experimental animals under conditions of weightlessness are the loss of
skeletal mass and attendant hypercalciuria. These clearly result from some
disruption in the balance between bone formation and bone resorption (i.e.
remodelling) which appears to be due to a decrease in the functions of the
osteoblast. In the studies funded by this project, the clonal osteoblastic
cell line, UMR 106-01, has been used to investigate the regulation of
collagenase and tissue inhibitors of metalloproteases (TIMPs). This project
has shed light on the comprehensive role of the osteoblast in the remodel-
ling process, and, in so doing, provided some insight into how the process
might be disrupted under conditions of microgravity.
Cloning and Regulation of Rat TIMP-2. Together with Dr. John Jeffrey, the
rat collagenase inhibitory activities secreted by the UMR 106-01 cells were
purified and shown to be two tissue inhibitors of metalloproteinase (TIMP)
molecules of 20 and 30 kDa. Amino-acid sequence of the inhibitor molecules
from medium conditioned by the rat UMR cells showed that one of these is
clearly homologous to TIMP-I in other species. The second, and predominant,
inhibitor molecule produced by UMR 106-01 cells has been identified as rat
TIMP-2, by virtue of its identity to human TIMP-2 in the amino terminal
region. We screened a rat genomic library with several different degenerate
oligonucleotide probes synthesized to known sequences of rat TIMP-2 and
obtained a positive clone. However, in addition, we obtained a human TIMP-2
cDNA which successfully hybridized to RNA from UMR cells. This indicated
that there was enough sequence homology between the two sequences to use it
as a probe to clone rat TIMP-2. We used this to screen our UMR cDNA library
and obtained numerous positive clones. These were then sequenced and
confirmed that we have obtained a rat TIMP-2 clone which has 98% amino acid
sequence identity with human TIMP-2.
Parathyroid hormone (PTH), at 10 .8 M, stimulated an approximately 2
fold increase in both the 4.2 kb and 1.0 kb transcripts over basal levels
in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2
transcripts was not affected by the inhibitor of protein synthesis,
cycloheximide (10 .5 M), suggesting a primary effect of the hormone. This is
in contradistinction to regulation of interstitial collagenase by PTH in
these same cells. Nuclear run-on assays demonstrated that PTH caused an
increase in TIMP-2 transcription which paralleled the increase in message
levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to
act through a signal transduction pathway involving protein kinase A (PKA)
since the increase in TIMP-2 mRNA was reproduced by treatment with the cAMP
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https://ntrs.nasa.gov/search.jsp?R=19970025580 2020-06-16T01:21:12+00:00Z
analogue, 8-bromo-cAMP (8BrcAMP, 5 x 10 .3 M). The protein kinase C (PKC)
and calcium pathways do not appear to be involved due to the lack of effect
of phorbol 12-myristate 13-acetate (PMA, 2.6 x 10 .6 M) and the calcium
ionophore, ionomycin (Iono, i0 "z M), on TIMP-2 transcript abundance. In
this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by
PTH are similar. However, we conclude that since stimulation of TIMP-2
transcription is a primary event, the PKA pathway must be responsible for a
direct increase in transcription of this gene.
Regulation of Collagenase Gene We demonstrated that stimulation of
collagenase by PTH involves a substantial increase in collagenase mRNA via
transcription. We have also concluded that this effect occurs primarily
through the cAMP-dependent pathway in UMR cells and requires the expression
of other genes. Our next goals were to identify the regulatory regions of
the rat collagenase gene. To do this, we obtained a genomic clone contain-
ing 6.5 kbp of sequence 5' of the transcriptional start site. This region
has been subcloned upstream of the reporter gene, chloramphenicol acetyl
transferase (CAT) and processive deletions made to determine the minimum
amount of sequence necessary for PTH induction. So far, PTH regulation is
maintained even with deletion to only 148 bases of sequence upstream of the
transcriptional start site. The largest of these constructs has been
transfected stably into UMR cells and will be characterized. These cells
could be used in flight experiments to assess changes in transcriptional
rate of the collagenase gene under microgravity conditions.
To assess the signal transduction pathway involved in PTH regulation
of collagenase gene expression, we investigated changes in immediate early
gene expression after PTH treatment. We demonstrated that c-fos and c-jun
mRNA levels are increased 20-fold and 2-5-fold 30 min after addition of
PTH. These effects precede the changes in transcription of collagenase
suggesting that these transcription factors may be the proteins which
induce transcription of the collagenase gene.
We have also examined regulation of collagenase in UMR cells by a
series of prostanoids as well as retinoic acid. Of the former, PGE 2 was the
most effective, reflecting the likelihood that the cAMP pathway is a major
regulator of expression of this gene in UMR cells. Retinoic acid also
stimulated collagenase expression by UMR cells. In both cases, the agents
increased transcription of the MMP-I gene and protein synthesis was
required for their effects. Thus, all the hormones investigated, so far,
appear to regulate collagenase by a secondary response pathway requiring
expression of some other gene.
Bone Cell Research (BCR) Experiment on Spacelab-Japanese (SL-J) Mission
We were recruited to Spacelab-J in November, 1990, and asked to test
whether my rat osteoblastic cells, UMR 106-01, would adhere and proliferate
normally in the NASDA cell culture hardware for the SL-J mission. After
many preliminary growth experiments, we found that with some manipulations
cells are viable, will grow and generally appear normal throughout the
period which was planned for space flight in this hardware.
Many other pre-flight experiments were also conducted in a very short
time. For instance, to alleviate the problem of lack of CO 2 in the Spacelab
incubator, we tested, and found suitable, a C02-independent medium from
Gibco. The cells were also tested to determine whether they would resist
the effects of vibration due to lift-off and showed no problems. We also
demonstrated that the hormone we were to use, PTH-related peptide, is quite
stable to freezing, thawing and incubation at 4°C for an extended period of
time. We also examined the stability of collagenase in medium at various
temperatures and resolved that the enzyme remains unchanged at 15°C for 4
days, while at 25°C, the enzyme is stable for 24 and 48 h, but is degraded
by incubation at this temperature for 72 h. These criteria established our
limitations for storage of our collected media samples on the Shuttle. Many
ELISA assays were conducted to test each of these parameters. In May, 1992,
Cheryl Johanns, my technician, was able to participate in a Science
Simulation at KSC which involved handover and scrub turnaround procedures.
It was only at this time that we learned the protocol for cleaning and
seeding the chambers from the Japanese investigators. Before this, we had
only been provided with very limited written notes on the use of the
chambers which had led to us developing procedures which may have not been
optimal.
At about the same time, the flight chambers were sent to us to bio-
condition and were used in a full simulation in the lab. We also sent
chambers with growing cells for a crew simulation in which media exchange
and photography was done. In the lab we did numerous photography sessions
to determine the methods that should be followed for good photomicroscopy
of the cells.
Launch was set for September ii, 1992. The launch was only a day late
(with plenty of notice) and most of the flight went smoothly. The Shuttle
landed at KSC which meant we received our samples within three hours of
landing and they were in very good condition. The limitations on the amount
of flight-certified hardware led to us not being able to conduct the best
ground controls. We conducted further ground control experiments in the
same flight chambers as were used on SL-J since the ones performed during
SL-J were not in optimal hardware. There was a misinterpretation by one of
the astronauts of the instructions on the first medium exchange which led
to many mitotic cells being sloughed off the chamber surface. This resulted
in far less cells being present than we anticipated. This has made it
difficult to measure collagenase produced in the medium since the cell to
medium ratio is so low. However, we were able to concentrate the media
before assaying the enzyme by our ELISA.
The photos taken by the astronauts are extremely good and we think
there are slight differences in the morphology of the flight cells compared
to our ground controls. Overall, we would have to conclude that this
hardware has severe limitations. The large size limits the number of
chambers which can be tested and the syringes are not ideal for media
exchanges. Also, the surface must be treated for use by various cells.
Our general experience of a flight experiment is that more time is needed
between being recruited to a mission and flight so that best use of the
hardware can be determined sooner, also complete training of the astronauts
needs to be accomplished.
We were unable to measure rat TIMPs by an ELISA since the affinities
of the antisera we obtained were insufficient and also cross-reacted with
bovine TIMPs (abundant in fetal bovine serum, FBS). The alternative method
would be to measure the TIMPs by a functional assay but this was also not
possible since the BCR media samples contained 10% FBS. This problem would
be exacerbated by concentration of the media 50-fold.
In conclusion, there appear to be some possible morphological changes
in the cells under microgravity but we are unable to determine whether
these are due to microgravity or the conditions of lift-off and/or vibra-
tion. This would require an on-board l-g centrifuge control to assess this.
Expression of Collagenase in Normal Differentiating Osteoblasts
In collaboration with Drs. Jane Lian and Gary Stein, we have found
that collagenase is expressed in normal differentiating rat osteoblasts.
Expression of collagenase is greatest in the most differentiated cells at a
time of greatest formation of mineralized nodules. We have now examined in
more detail this appearance of collagenase in normal, mineralizing,
differentiating osteoblastic cultures. Collagenase is just detectable in
the early part of the culture, 7 and 14 days, but then starts to increase
at day 21 reaching high levels at 28 days, then declining at later ages (35
days). This profile is similar to the changes in osteopontin and
osteocalcin in this type of differentiating osteoblast system. Thus,
collagenase is expressed in these cultures as a late-differentiation gene
which likely is involved in matrix maturation.
We have examined the effect of mineralization of the matrix on the
expression of collagenase in these cultures. Basal levels of collagenase
were expressed in both sets of cultures but only the group with enhanced
mineralization demonstrated the late differentiation surge in collagenase.
We have also measured cell growth under these two sets of conditions and
the cells cultured in MEM continue to proliferate throughout the culture
period, developing layers of cells; the cells in BGJ b medium cease prolif-
eration• Thus, several factors could be responsible for the stimulation of
expression of collagenase: The cessation of proliferation could be respon-
sible for the increase in collagenase expression. However, this seems
unlikely because, proliferation ceases at 7 days but the greatest increases
in collagenase occur 14 days later. Secondly, the increase in mineraliza-
tion could initiate the upturn in collagenase mRNA levels. However, like
proliferation, this also is initiated as soon as the cell are transferred
to the mineralization medium. What is striking is that the increase in
collagenase generally occurs as alkaline phosphatase is declining. Presum-
ably, one set of transcription factors is responsible for elevation of
alkaline phosphatase and levels of these, in turn, may decrease in line
with decrease in alkaline phosphatase expression. This could be a signal
for production of another set of transcription factors or the decline
itself may be a signal for increased collagenase transcription.
We have also shown that collagenase in these cultures can be stimu-
lated by PTH treatment. However, PTH cannot stimulate expression until
basal expression is initiated.
Publications Resulting from the Project Since Last Competitive Review
•
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•
Puccinelli, J.M., Omura, T.H., Strege, D.W., Jeffrey, J.J. and Par-
tridge, N.C. A serum factor promotes collagenase synthesis by an
osteoblastic cell line. J. Cell. Physiol., 147, 505-513, 1991.
Roswit, W.T•, McCourt, D.W., Partridge, N.C. and Jeffrey, J.J. Purifi-
cation and sequence analysis of two rat tissue inhibitors of metallop-
roteinases. Arch. Biochem. Biophys., 292, 402-410, 1992.
Scott, D.K., Brakenhoff, K.D., Clohisy, J.C., Weaver, W.R., Kahn, A.J.
4
.So
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o
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So
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ii.
12.
13.
14.
15.
16.
and Partridge, N.C. Regulation of an H-ras-related transcript by
parathyroid hormone in rat osteosarcoma cells. Mol. Endocrinol., 6,
1425-1432, 1992.
Clohisy, J.C., Scott, D.K., Brakenhoff, K.D., Quinn, C.O. and Par-
tridge, N.C. Parathyroid hormone induces c-fos and c-Sun mRNA in rat
osteoblastic cells. Mol. Endocrinol., 6, 1834-1842, 1992.
Scott, D.K., Brakenhoff, K.D., Clohisy, J.C., Quinn, C.O. and Par-
tridge, N.C. Parathyroid hormone induces transcription of collagenase
in rat osteoblastic cells by a mechanism utilizing cAMP and requiring
protein synthesis. Mol. Endocrinol., 6, 2153-2159, 1992.
Omura, T.H., Noguchi, A., Quinn, C.O., Jeffrey, J.J. and Partridge,
N.C. Receptor-mediated binding of collagenase to osteoblasts. In
"Calcium Regulating Hormones and Bone Metabolism" (eds. D.V. Cohn, C.
Gennari and A.H. Tashjian Jr.), pp. 356-360, 1992.
Shalhoub, V., Conlon, D., Tassinari, M., Quinn, C., Partridge, N.,
Stein, G.S. and Lian, J.B. Glucocorticoids promote development of the
osteoblast phenotype by selectively modulating expression of cell
growth and differentiation associated genes. J. Cellular Biochem., 50,
425-440, 1992.
Martin, T.J., Ng, K.W. and Partridge, N.C. Properties of
transplantable osteogenic sarcoma and derived cell clones. In "Fron-
tiers of Osteosarcoma Research" (ed. J.F. Novak and J.H. McMaster)
Hogrefe and Huber Publishers, Seattle, WA, pp.247-255, 1993.
Partridge, N.C., Scott, D.K., Gershan, L.A., Omura, T.H., Burke, J.S.,
Jeffrey, J.J., Bolander, M.E. and Quinn, c.o. collagenase production
by normal and malignant osteoblastic cells. In "Frontiers of Osteosar-
coma Research" (ed. J.F. Novak and J.H. McMaster) Hogrefe and Huber
Publishers, Seattle, WA, pp.269-276, 1993.
Clohisy, J.C., Scott, D.K., Partridge, N.C. and Quinn, C.O. Parathy-
roid hormone stimulation of matrix metalloproteinase I gene expression
in rat osteosarcoma cells. In "Frontiers of Osteosarcoma Research"
(ed. J.F. Novak and J.H. McMaster) Hogrefe and Huber Publishers,
Seattle, WA, pp. 531-535, 1993.
Partridge, N.C., Bloch, S.R. and Pearman, A.T. Signal transduction
pathways mediating parathyroid hormone regulation of osteoblastic gene
expression. J. Cellular Biochem., 55, 321-327, 1994.
Cook, T.F., Burke, J.S., Bergman, K.D., Quinn, C.O., Jeffrey, J.J. and
Partridge, N.C. Cloning and regulation of rat tissue inhibitor of
metalloproteinases-2 in osteoblastic cells. Arch. Biochem. Biophys.,
311, 313-320, 1994.
Clohisy, J.C., Connolly, T.J., Quinn, C.O. and Partridge, N.C.
Prostanoid-induced expression of matrix metalloproteinase-i mRNA in
rat osteosarcoma cells. Endocrinology, 135, 1447-1454, 1994.
Connolly, T.J., Clohisy, J.C., Partridge, N.C. and Quinn, C.O.
Retinoic acid stimulates interstitial collagenase mRNA in osteosarcoma
cells. Endocrinology, 135, 2542-2548, 1994.
Partridge, N.C., Lorenz, T.C., Morey-Holton, E.R., Durnova, G.,
Gershan, L.A., Jeffrey, J.J., Kaplansky, A.S. and Quinn, C.O. Immuno-
histochemistry of collagenase in calvariae of rats flown on
Cosmos-2044. NASA Technical Memorandum 108802, 213-223, 1994.
Partridge, N.C., Walling, H.W., Bloch, S.R., Omura, T.H., Chan, P.T.,
Pearman, A.T. and Chou, W.-Y. The regulation and regulatory role of
_5
17.
collagenase in bone. Critical Reviews in Eukaryotic Gene Expression,
6, 15-27, 1996.
Koe, R., Clohisy, J.C., Tyson, D.R., Pulumati, M.R., Cook, T.F. and
Partridge, N.C. Parathyroid hormone versus phorbol ester stimulation
of activator protein-i gene family members in rat osteosarcoma cells.
Calcif. Tiss. Int., 61, 52-58, 1997.
6


Skeletal Collagen Turnover by the Osteoblast

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