Bioscience Reports, Vol. 13, No. 3, 1993
Tetracyclines Modulate Cytosolic C a 2+
Responses in the Osteoclast Associated with
" C a 2+ Receptor" Activation
Christopher M. R, Bax, 1 Vijai S. Shankar, ~ A. S. M.
Towhidul Alam, ~ Bridget E. Bax, ~ Baljit S. Moonga, ~
Christopher L.-H. Huang, 2 Mone Zaidi, ~'4 and Barry R.
Rifkin 3
Received February 15, 1993
We report the effects of tetracycline analogues on cytosolic Ca2+ transients resulting from application
of ionic nickel (Ni2+), a potent surrogate agonist of the osteoclast Ca2+ "receptor". Preincubation
with minocycline (1 rag/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline
(CMT-1) (1 or 10 mg/1), resulted in a significant
attenuation of the magnitude of the cytosolic [Ca2+ ]
.z+
response to an application of 5 mM-[Ni ]. Preincubation with doxycycline (1 or 10mg/l) failed to
produce similar results. In addition, application of minocycline alone (0.1-100rag/I) resulted in a
3.5-fold elevation of cytosolic [Ca2+]. The results suggest a novel action of tetracyclines on the
osteoclast Ca2+ "receptor".
KEY WORDS: tetracycline; osteoclasts; cytosolic Ca2+.
INTRODUCTION
There is clear evidence that tetracyclines inhibit osteoclastic b o n e resorption
(1, 2). In addition, the drugs e n h a n c e osteoblast function, but inhibit the activity
of the secreted colJagenase (3, 4). T h e s e actions have m a d e the tetracycline family
potentially attractive for the t r e a t m e n t of certain osteopenias ( 3 , 5 ) . Also of
interest is the suggestion that the anti-osteopenic effects of tetracyclines m a y be
i n d e p e n d e n t of their anti-microbial activity (3). Tetracyclines have recently b e e n
shown also to attenuate cytosolic [Ca 2+] signals resulting f r o m an elevated
extracellular [Ca 2+] ( 6 - 8 ) , suggesting possible actions of the drugs on specific
pathways that m a y be i m p o r t a n t in the control of osteoclast function (9). W e n o w
Division of Biochemical Medicine, St. George's Hospital Medical School, London, SWI7 ORE, UK.
2 Physiological Laboratory, Cambridge University, Cambridge, CB2 3EG, UK.
3 Department of Oral Medicine and Pathology, New York University, New York, New York, USA.
4 To whom correspondence should be addressed.
169
0144-8463/93/0600-01695117.00/(I 9 1993 Plenum Publishing Corporation
Bax et
170
al.
further examine the pharmacological basis of tetracycline action on the cellular
activation pathway which has been shown in a series of recent reports to be
triggered by ionic nickel (Ni2+), a surrogate Ca 2§ "receptor" agonist (10). A
tetracycline of particular interest was a chemically modified analogue, 4dedimethylaminotetracycline (CMT-1). This analogue is known not to possess
appreciable anti-microbial activity, but retains its ability to inhibit osteoblastic
collagenase activity and osteoclastic bone resorption (11).
MATERIALS AND METHODS
Osteoclasts were mechanically disaggregated from neonatal rat long bones in
Medium 199 (ICN Flow, U.K.) buffered with N'-2-hydroxyethyl piperazine-N'-2ethane sulphonic acid (HEPES) (Sigma, Dorset, U.K.) and supplemented with
foetal calf serum (10% v/v) (Gibco, Uxbridge, Middx., U.K.). The supernatant
was dispersed onto glass coverslips; whereas osteoclasts rapidly sedimented and
attached to the substrate, other cells could be washed away (12). [Ca2+]i was
measured in single isolated osteoclasts by a dual excitation microspectrofluorimetric method using fura 2, a Ca2+-sensitive fluorescent dye (13). The microspectrofluorimeter was previously constructed from an inverted phase-contrast
microscope fitted with epifluorescence using a 100W xenon lamp source
(Diaphot, Nikon UK Ltd., Telford, U.K.). Fura 2-loaded osteoclasts were
exposed alternately to excitation wavelengths of 340 and 380 nm by means of a
microcomputer-driven filter wheel. The emitted fluorescence passed through a
dichroic mirror (400 nm), and the transmitted light was filtered at 510 nm. The
fluorescent beam was finally directed to the sideport of the microscope to which a
variable aperture, shutter and photomultiplier tube (PM28B, Thorn EMI,
London, U.K.) were attached. Single photon currents from the tube were
converted to 5 volt, 25 nanosecond transistor-transistor logic (TTL) pulses and
counted by a dual photon counter (Newcastle Photometric Systems, Newcastleupon-Tyne, U.K.). Photon counts in each channel were recorded approximately
each second in a IBM microcomputer and the ratio of emitted intensities due to
excitation at 340 and 380 nm (F340/F380) calculated and displayed. Osteoclasts,
settled on glass coverslips (22 mm, grade 0; Chance Propper, U.K.) were loaded
with 10/~M acetoxymethyl ester of fura 2 (fura 2-AM) by incubating in
serum-free medium for 30 minutes at 37~ The coverslips were then transferred
to a bath on the stage of the microspectrofluorimeter and exposed to minocycline
or NiC12 by the manual pipetting of solutions prewarmed to 37~ In other
experiments, osteoclasts were pre-treated for 1 hour with minocycline, doxycycline (Sigma) and CMT-1, before being loaded with the fluorochrome. The
solution temperature was kept at 37~ by means of a thermostatically controlled
heater, and the volume was maintained by the use of a constant-level vacuum
device. [Ca2+]i was estimated by using an intracellular calibration protocol as
described earlier (13).
Tetracycline Effects on Osteoclast Function
171
RESULTS
Effect of Tetracyclines on Osteoclast Cytosolic [Ca z+]
Application of minocycline elicited a modest, but consistent rise i n [Ca2+]i ,
which was not observed when vehicle was added. E a c h [Ca2+]i response was
followed by a spontaneous decrease in [Ca2+]i to baseline. At the concentrations
tested (0.1, 1, 10 or 100mg/1), a rise of 1.7-, 3.1-, 2.7- and 3.2-fold respectively,
above basal [Ca2+]i w a s observed. The elevation o f [Ca2+]i was marginally
significant at minocycline concentrations of 0.1 and 1.0 mg/1 (0.06 > p > 0.05),
and significant at the higher concentations (p < 0.05). However, the magnitude of
the [CaZ+]i response to minocycline was not concentration-dependent (p > 0 . 3 ,
1-way anova; n = 5 to 10). The acute effect of doxycycline and CMT-1 on [Ca2+]~
could not be assessed due to the fluorescence artefacts.
Effect of Ni z+ Pretreatment on Minocycline-induced Cytosolic [Ca z+] Elevation
Application of 5 mM-[Ni 2+] to isolated rat osteoclasts resulted in a rapid
monophasic elevation of cytosolic [Ca2+]i rising from a resting level of 36 + 4 nM
to a peak of 362 + 5 0 n M (mean + standard error of mean; p < 0 . 0 0 1 using
Student's paired t-test, n = 21). A short conditioning, 2- to 4-minute exposure to
5 mM-[Ni 2+] failed to affect the [Ca2+]i response to a subsequent application of
minocycline. Thus, minocycline-induced A[Ca2+]i (peak [Ca2+]i minus pre-peak
[Ca~+]~, + standard error of mean; nM; n = 5 or 6) without Ni 2+ pre-treatment
(49+ 19 and 4 2 ~ 10 respectively, for 1 and 10mg/1-[minocycline]) was not
significantly different from that following prior exposure to Ni 2+ (78 :]z 31 and
40 + 6 respectively, for the two concentrations of minocycline) (p > 0.5, in each
case).
Table 1. The effect of brief pretreatment of
osteoclasts with minocycline on Ni2+-induced
elevation of cytosolic Ca 2+ ([Ca2+]i)
[Minocycline]
0.1
1-0
10
100
A[Ca2+]i
422 •
372 •
798 +
207 •
235
142
346
73.0
n
5
4
6
6
Results are expressed as mean change in
[Ca2+]i2+ (A[Ca2+]i; peak [Ca2+]i minus" pre-peak
[Ca ]i) + standard error of mean, nM, in response to the application of 5 mM-[Ni2+]. [Minocycline], rag/l; n, number of cells. Student's
unpaired t-test was used to compare treatment
groups with the control response to 5 mM[Ni 2+] obtained in the absence of minocycline
(327 + 51 riM, n = 21)(p > 0.2)
172
Bax et al.
5 mM-[Ni 2§
9
400
~.
k
3oo
~
%
O
<1
(a)
.~..
200
"
"~-
1 O0
~.~D,:.~.~,~
~
"-..-._Z_
---r
0
i
i
0
100
i
i
200
T i m e (sec)
300
Fig. l. Representative traces showing the changes in
the cytosolic free Ca 2+ concentration (A[Ca2+]i) (nM)
in single rat osteoclasts in response to application of
5 mM-[Ni2+] to cells that had been preincubated for 1
hour
in
vehicle
(Medium
199)
(a),
4dedimethylaminotetracycline (CMT-1) (1 nag/l) (b) or
minocycline (1 mg/1) (c).
2. The effects of 1-hour pretreatment with doxycycline, 4-dedimethylaminotetracycline (CMT-1) or
minocycline, on Nie+-induced elevation of cytosolic
[Ca 2+] ([Ca2+]i) in single fura 2-loaded osteoclasts.
Table
Treatment
[Doxycycline]
1
10
[CMT-1]
1
10
[Minocycline]
1
10
A[Ca2+]i
n
p
386 5:159
290 + 112
6
6
>0.70
>0.70
129 • 6
102.4- 16
3
3
<0.06
<0.05
86 • 25
6
215 • 40
6
<0.01
>0.10
Results are expressed as mean change in cytosolic free
calcium (A[Ca2+]i) (peak [Ca2+]i minus pre-peak
[Ca2+]i ) + standard error of mean, nM, in response to
the application of 5 mM-[Ni2+]. [Tetracycline], mg/1; n,
number of cells. The Student's unpaired t-test has been
used to compare responses obtained in tetracycline
pre-treated osteoclasts with control responses to 5 mM[Ni 2+] obtained without pretreatment with the respective
tetracyclines (327 + 51 nM) (n = 21). When cells were
incubated with CMT-I for prolonged periods, there was
evidence, in some cells, of an effect of the compound on
dye fluorescence, rendering the data from such cells
invalid
Tetracycline Effectson Osteoclast Function
173
Effect of Tetracycline Pretreatment on NiZ+-induced Cytosolic [Caz+]
Elevation
In a different set of experiments, osteoclasts were exposed to minocycline for
between 2 and 4 minutes before the application of 5 mM-[Ni2+]. The resulting
changes in [Ca2+]i were compared to control Ni2+-induced [Ca2+]i responses. The
results are summarised in Table 1. Pretreatment with 0.1, 1.0, 10 or 100mg/1[minocycline] resulted in no apparent changes in the magnitude of Ni2+-induced
[Ca2+]i elevation (p > 0.3, 1-way anova). Further experiments were performed in
which ceils were pretreated for 1 hour with minocycline, doxycycline or CMT-1
prior to application of 5 mM-[Ni 2+] (Figs lb and lc). Minocycline (1 mg/l) or
CMT-1 (1 and 10mg/I) were found to result in a significant reduction in the
magnitude of the NiZ+-induced [Ca2+]i response, whilst doxycycline (1 or 10 mg/l)
failed to attenuate the [Ca2+]i response to 5 mM-[Ni 2+] (Table 2).
DISCUSSION
We have shown here that application of minocycline to isolated osteoclasts
results in a significant elevation of cytosolic [Ca 2+] at concentrations (0.1 to
100 mg/1) well within the accepted therapeutic plasma concentration range (1 to
4 rag/l) (14). This effect may explain the reported anti-bone resorptive effect of
minocycline (11). It is now well known that physiological and pharmacological
agents that elevate cytosolic [Ca 2+] inhibit bone resorption using this mechanism.
These include the hormone calcitonin (15), elevated extracellular [Ca 2+] (6, 7),
perchlorate ions (16), verapamil (17) and ionomycin (8). Furthermore, it is likely
that the observed elevation of cytosolic [Ca2+] results directly from a partial
agonist effect of minocycline on the osteoclast Ca 2+ "receptor". This would be
consistent with earlier observations showing that minocycline and doxycycline can
reduce cytosolic [Ca 2+] responses to elevated extracellular [Ca2+], but not to
calcitonin (9).
We obtained further evidence for the action of tetracyclines on the osteoclast
Ca 2+ "receptor" by pre-incubating osteoclasts with the drugs followed by
exposure to Ni 2+, a known agonist of the Ca 2+ "receptor". The hypothesis was
that, if tetracyclines interact at the same site as Ni 2+, then they may attenuate the
[Ca2+]i response to application of the cation. Thus, when osteoclasts were
incubated for 1 hour with minocycline (1 mg/1) or with CMT-1 (1 or 10mg/l)
there was a significant reduction in the magnitude of the cytosolic [Ca 2+] response
to 5 mM-[Ni 2+] application. This was not seen with doxycycline (1 or 10 mg/l).
However, since doxycycline inhibits osteoblast collagenase and parathyroid
hormone-induced bone resorption at least as well as minocycline and CMT-1, it
remains to be determined whether tetracyclines have an additional mode of
action. Furthermore, the effects of minocycline and CMT-1 were time-dependent.
Thus, a brief exposure of the cells to minocycline did not affect the response to
Ni 2+. This would appear to argue against direct competitive inhibition and may
suggest an alternative action on the Ca 2+ receptor, such as the causing of a
conformational change in the cation-binding site.
174
Bax et al.
Any substance which acts at the osteoclast Ca 2+ receptor and mimics the
action of Ca 2+ would be of interest therapeutically in osteoclastic bone disease,
including osteoporosis. Our suggestion that the non-antibiotic analogue from the
tetracycline family, CMT-1, can interact with the C a 2+ "receptor" may result in
novel tetracyclines being assessed for osteoclast-inhibitory activity. A convenient
and easy screening procedure for potential osteoclast-inhibitory activity would be
a [CaZ+]i-measuring system as described above. Although we have established
that minocycline is a partial Ca 2+ "receptor" agonist, a tetracycline having full
agonist activity would be of considerable interest therapeutically.
ACKNOWLEDGEMENTS
The study was supported in part from the Arthritis and Rheumatism Council,
U.K., (MZ), Medical Research Council, U.K., (MZ), Leverhulme Trust, U.K.,
(MZ) and National Institutes of Health, U.S.A., (DEO9576) (BRR). ASMTA
was a recipient of the Overseas Research Award (U.K.).
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