B-dependent gene regulation
Department of Pathology, Duke University Medical Center, Durham, North Carolina
Correspondence: Salvatore V. Pizzo, M.D., Ph.D., Professor and Chairman of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710. E-mail: Pizzo001{at}mc.duke.edu
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B and CREB transcription factors and the proto-oncogenes
c-fos and c-myc were also elevated
significantly. Intracellular Ca2+ chelation
blocked the effect of BeF2. We conclude that
BeF2 at low concentration exerts its mitogenic effects in
peritoneal macrophages by elevating
[Ca2+]i, which triggers the
activation of p21ras-dependent MAPK signaling
cascades.
Key Words: mitogenesis • MAPK regulation • AlFn • CREB
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subunit of heterotrimeric G
proteins in the guanosine 5'-diphosphate (GDP)-bound form can be
activated by AlFn or BeFn
[14
15
16
17
18
]. It is thought that the metal ion-fluoride
complexes mimic the terminal phosphate of guanosine 5'-triphosphate
(GTP), such that the structure of the G-GDP-AlFn (or
BeFn) complexes resembles that of the GTP-bound form of the
protein [14
15
16
17
18
]. Activation of purified G proteins by
metal ion-fluoride complexes results in a change in the conformation of
the G subunit and release of Gß
, which promotes association of
the G subunits with effector molecules downstream of the G protein
[14
15
16
17
18
]. The lifetime of the interaction of these metal
ion-fluoride complexes with G proteins, and hence of the activated G
proteins, is influenced by several effectors including
GTPase-activating proteins (GAPS) [16
17
18
]. Mitogen activated protein kinases (MAPKs) are a family of Ser/Thr kinases that mediate intracellular signal transduction in response to various stimuli. MAPKs are involved in cellular stress responses and apoptosis [19 20 21 22 23 ] and are themselves activated by phosphorylation [19 20 21 22 ]. Three major MAPK families have been identified: the extracellular signal-regulated kinases (ERK1/2), the stress-activated protein kinases known as c-jun NH2-terminal kinases (JNK), and the p38 MAPKs [19 20 21 22 ]. Ras-dependent activation of MAPK kinases that activate ERK, (MEK)1 and 2, leads to activation of ERK1/2 [19 20 21 22 ]. This pathway provides a common route by which signals from different growth-factor receptors converge to activate major transcription factors such as activated protein-1 (AP-1) [19 20 21 22 23 ]. Ras also activates JNK, which plays a role in regulating AP-1 activity [24 ]. Variations in [Ca2+]i are a major determinant in the regulation of the Ras/MAPK pathway [19 20 21 22 23 ]. G-protein-dependent activation of phospholipase C results in hydrolysis of membrane phosphoinositides generating inositol (1,4,5)trisphosphate (IP3) and diacylglycerol (DAG) [25 , 26 ]. IP3 raises [Ca2+]i by binding to specific receptors on the endoplasmic reticulum (ER), thus mobilizing membrane-sequestered Ca2+ [25 , 26 ]. DAG is required for activating protein kinase C (PKC) [25 , 26 ].
Recently, we have observed that exposure of murine peritoneal
macrophages to beryllium chloride (BeCl)2 increased
[Ca2+]i levels in an
IP3-independent manner and enhanced
[3H]thymidine uptake in a calcium-dependent manner. In
this study, we demonstrate that exposure of macrophages to
BeFn alters calcium homeostatis and mitogenesis in a manner
significantly different than that observed with BeCl2
treatment [27
]. We show that in macrophages,
BeFn at low concentration (<10 nM) increase
[3H]thymidine uptake and cell number about twofold.
Concomitantly, we observed a similar increase in the levels of
phosphorylated MEK1, ERK1, p38 MAPK, and JNK. Inhibition of these
kinases by specific inhibitors significantly attenuated the
BeFn-induced effects described above. BeFn also
affected the levels of the transcription factors, nuclear factor-B
(NF-B) and cyclic AMP response element-binding (CREB) protein. The
effects of BeFn are dependent on
[Ca;2+]i levels, which at least in part are a
consequence of IP3 generation. BeFn also
triggered Ca2+ uptake by L-type channels.
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B protein were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Other
chemicals used were procured locally and were of the highest available
purity.
Determination of macrophage cell number after exposure to
BeFn
Because increased DNA synthesis is normally associated with an
increase in total cellularity, the number of macrophages before and
after overnight exposure to varying concentrations of BeF2
was determined. Peritoneal macrophages were harvested from C57BL/6 mice
three days after intraperitoneal injection of thioglycollate as
described previously [29
] and were allowed to adhere in
six-well plates in RPMI-1640 medium containing 5% fetal bovine serum
(FBS) for 2 h. The adhered cells were scraped carefully,
centrifuged at 1200 rpm for 5 min, and suspended in 1.5 ml RMPI-1640
medium containing 0.2% bovine serum albumin (BSA) and 0.5 ml aliquots
(2x105 cells) pipetted into 15 ml siliconized
polypropylene tubes. To the respective tubes, a specified concentration
of BeF2 was added, the contents mixed gently, and the tubes
incubated overnight as above. After overnight incubation, 10 µl
trypan blue solution was added to each tube, the tubes were gently
shaken during incubation for 2 min, and a 10 µl aliquot was used for
counting the number of cells by hemocytometers. In experiments where
the modulation in cell numbers of BeF2-exposed macrophages
by [Ca2+]i, MAPKs, or Ras was
examined, the macrophages were treated with the intracellular calcium
chelator BAPTA/AM (10 µM/30 min); U0126 (1 µM/10 min), a specific
inhibitor of MEK1; PD98059 (50 µM/90 min), a specific inhibitor of
ERK; or SB203580 (25 µM/30 min), a specific inhibitor of p38 MAPK, or
manumycin A (25 µM/60 min), a specific inhibitor of
farnesyltransferase [28
]. The latter enzyme is required
for activation of p21ras [28
]. These agents
were added to their respective tubes, which were incubated for the
specified time before adding BeF2 (2 nM). Cell numbers were
determined as described above.
[3H]Thymidine uptake by BeFn-exposed
macrophages
Murine peritoneal macrophages (4x105 cells/well in
48-well plates) harvested as above were allowed to adhere for 2 h
in RPMI-1640 medium containing 5% FBS, penicillin, streptomycin, and
glutamine at 37°C in a humidified CO2 (5%) incubator.
The monolayers were washed twice with Hanks’ balanced salt solution
containing 10 mM HEPES, 3.5 mM NaHCO3, pH 7.2 (HHBSS). A
volume of the above RPMI medium was added, except that sera was
replaced with 0.2% BSA. [3H]Thymidine was then added,
followed by varying concentrations of BeF2 or NaF. The
cells were incubated overnight in a humidified CO2 (5%)
incubator. The incubations were terminated by aspirating the medium and
washing macrophages twice, first with 5% trichloroacetic acid (15
min/4°C) and then thrice with HHBSS. The monolayers were lysed with 1
N NaOH, and an aliquot was used for liquid scintillation counting and
protein estimation [30
].
The role of [Ca2+]i levels
and the activity of PKC, phosphatidylinositol-specific phospholipase C
(PI-PLC), tyrosine kinases, and MAPKs in BeFn-induced
[3H]thymidine uptake
In experiments that examined effects of modulating
[Ca2+]i levels in
BeFn-induced [3H]thymidine uptake,
[Ca2+]i levels were modulated
with BAPTA/AM (10 µM/30 min) or nifedipine (5 µM/10 min). These
agents were added to individual wells, and the cells were incubated for
the specified time period before adding BeF2. In studies on
modulation of [3H]thymidine uptake, the respective
monolayers were preincubated with U73122 (4 µM/10 min), an inhibitor
of PI-PLC; chelerythrin (200 nM/10 min), an inhibitor of PKC; genestein
(20 µM/16 h), an inhibitor of tyrosine kinases, U0126(2 µM/10 min),
an inhibitor of MEK1; PD 98059 (50 µM/90 min), an inhibitor of ERK;
SB203580 (15 µM/30 min), an inhibitor of p38 MAPK; or manumycin A (10
µM/1 h), a farnesyltransferase inhibitor, before adding
BeF2 and [3H]thymidine. Details of
incubations and determination of cell lysate radioactivity were as
described above.
Measurement of IP3
Murine peritoneal macrophages were obtained as described above.
The cells were centrifuged, suspended in RPMI-1640 medium containing
12.5 units of penicillin/ml, 6.5 µg/ml streptomycin, 2 mM glutamine,
and 5% FBS, plated in six-well plates (4x106 cells/well
in six-well plates), and allowed to adhere for 2 h at 37°C in a
humidified 5% CO2 incubator. Monolayers were washed with
HHBSS, a volume of above-mentioned RPMI, except that serum was
substituted with 0.2% fatty acid-free BSA added, followed by the
addition of [3H]myoinositol (8 µCi/well), and the
monolayers were incubated overnight as described above. The monolayers
were washed with chilled HHBSS containing 10 mM LiCl, 1 mM
CaCl2, and 1 mM MgCl2 thrice, a volume of wash
buffer was added, and monolayers were preincubated for 3 min at 37°C
as above, before stimulating with BeF2 (2 nM) or with
aluminum chloride (10 µM)-sodium fluoride (50 mM). Other details of
fractionation and quantification of radioactivity of IP3
were performed as described previously [29
].
Measurements of changes in intracellular calcium levels in
BeFn-exposed macrophages
Changes in [Ca2+]i levels
in Fura-2/AM-loaded macrophages on exposure to BeF2 were
measured by digital imaging microscopy essentially as described
previously [29
]. Briefly, freshly harvested murine
peritoneal macrophages were suspended in RPMI-1640 medium containing
5% FBS, penicillin, streptomycin, and glutamine. They were then
allowed to adhere on glass coverslips and kept in 35-mm petri dishes at
37°C for 2 h in a humidified CO2 (5%) incubator.
The nonadherent cells were aspirated, and the monolayer was washed with
HHBSS, a volume of RPMI medium was added, and cells were incubated
overnight as above. Macrophages incubated overnight were loaded with
Fura-2/AM (4 µM/30 min at 25°C in dark) and washed thrice with
HHBSS, and a volume of HHBSS was added. The cells on coverslips were
exposed to BeF2, and changes in
[Ca2+]i in single cells were
quantified as described previously [29
]. The origin of
elevated levels of [Ca2+]i in
BeF2-exposed cells could result from its elevated release
from IP3-sensitive Ca2+ stores,
influx of extracellular Ca2+ via specific
channels, IP3-independent release from ER
Ca2+ stores, and/or inhibition of
[Ca2+]i extrusion. The
contribution of IP3-dependent release of
Ca2+ from ER calcium stores to the total
[Ca2+]i pool in
BeF2-exposed macrophages was analyzed by treating
Fura-2/AM-loaded cells with PI-PLC inhibitor U73122 (4 µM/10 min)
before adding BeF2 and measuring changes in Fura-2
fluorescence [31
]. The contribution of
BeF2-induced influx of Ca;2+ to the total
[Ca2+]i pool in
BeF2-exposed cells was assessed by treating the cells with
nifedipine (5 µM/10 min), specific blockers of L-type
Ca2+ channel [32
], or
flunarizine • HCl (5 µM/10 min), a selective blocker of
low-threshold T-type channel, before exposure to BeF2 and
measurement of changes in Fura-2 fluorescence.
Western blotting of phosphorylated MEK1, ERK1, p38 MAPK, and JNK in
macrophages exposed to BeFn
Freshly harvested peritoneal macrophages in RPMI-1640 medium
containing penicillin, streptomycin, glutamine, and 5% FBS were
allowed to adhere in six-well plates (3x106 cells/well)
for 2 h as above. The monolayers were washed twice with HHBSS, and
a volume of above-mentioned RPMI-1640 medium was added, except that the
serum was substituted with 0.2% BSA, and plates were incubated
overnight as above. The monolayers were washed twice, and a volume of
RMPI medium containing 0.2% BSA was added. The cells were then exposed
to buffer or BeF2 (2 nM/20 min) after adding the specific
inhibitors/modulators of MAPKs. The concentrations of these inhibitors
and the incubation time periods were as described above. The
incubations were terminated by aspirating the medium. The lysis of
cells, their electrophoresis, and Western immunoblotting were performed
according to the manufacturer’s instruction. In each case, equal
amounts of protein were used for electrophoresis. The detection of
phosphorylated MAPKs by enhanced chemifluorescence (ECF) and
quantification of their distribution were performed by PhosphorimagerTM
(Storm 860, Molecular Dynamics, Sunnyvale, CA).
Western blotting of c-fos, c-myc, CREB, and NF-B proteins in
macrophages exposed to BeFn
Freshly harvested peritoneal macrophages in RPMI-1640 medium
containing penicillin, streptomycin, glutamine, and 5% FBS were
allowed to adhere in six-well plates (3x106 cells/well)
for 2 h as above. The monolayers were washed twice with HHBSS, and
a volume of RPMI-1640 medium was added, except that the serum was
substituted with 0.2% BSA, and plates were incubated overnight as
above. The monolayers were washed, a volume of RPMI medium containing
0.2% BSA was added, and the cells were exposed to buffer or to
BeF2 (2 nM/20 min). The respective incubations were
terminated by aspirating the medium. The lysis of cells, their
electrophoresis, and Western immunoblotting were done according to the
manufacturer’s instruction. In each case, an equal amount of protein
was used for electrophoresis. The detection of immunoblots was
performed by ECF and quantification of their distribution by
PhosphorimagerTM (Storm 860, Molecular Dynamics).
Measurement of the effect of NF-B activation on
BeFn-induced [3H]thymidine incorporation
The involvement of the NF-B transcription factor in
BeFn-induced uptake was examined by incubating the cells
with two specific inhibitors of NF-B, namely BAY 11-7082 (10 µM/10
min) [33
] and PGA1 (25 µM/10 min)
[34
]. These inhibitors prevent agonist-induced
phosphorylation and degradation of IB, resulting in the inactivation
of NF-B activation, and hence, inhibit its translocation to nuclei
[35
36
37
]. These inhibitors were added to respective
monolayers and incubated for the specified time before adding
BeF2. Details of incubation and determination of cell
lysate radioactivity were the same as described above. Statistical
analysis of the data was performed by Student’s t-test, and
P values 
0.05 are considered significant.
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Figure 1. BeF2-induced cell division and [3H]thymidine
incorporation into peritoneal macrophages. (A) Effect of increasing
concentration of BeF2 on murine peritoneal macrophage cell
number after 24 h incubation. The cell numbers are mean ±
SE from two independent experiments counted in
quadruplicate. (B) Effect of increasing concentrations of
BeF2 on [3H]thymidine
incorporation into macrophages. Values are mean ± SE
from two experiments performed in quadruplicate and are expressed as
fmol [3H]thymidine incorporated/mg cellular protein. The
experimental data were significantly different at P <
0.005 compared with buffer-treated controls. (C) Effect of increasing
concentrations of NaF on [3H]thymidine incorporation in
macrophages. Values are mean ± SE from two
experiments performed in quadruplicate and are expressed as fmols
[3H]thymidine incorporation/mg cellular protein.
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The effect of BeFn exposure of macrophages on
[Ca2+]i
Exposure of macrophages to BeFn species increased
[Ca2+]i levels by two- to
threefold (Fig. 2 A
). The increase was largely in single, large spikes, sometimes
accompanied by smaller spikes. The effect of BeF2 is
concentration-dependent, and a maximal effect is seen at 2–5 nM.
Thereafter, responses declined and were essentially absent above 50 nM
concentrations of BeF2 (Fig. 2B)
. By contrast, macrophages
exposed to BeCl2 show an increase in
[Ca2+]i, which reaches a plateau at 100 nM,
and the response is sustained thereafter, up to a concentration of at
least 500 nM BeCl2 in the absence of IP3
synthesis [27
].
 View larger version (14K): [in a new window] |
Figure 2. Changes in [Ca2+]i in
Fura-2/AM-loaded macrophages exposed to BeF2. (A) A
representative cell from 100–150 cells from five individual
experiments each using 25–30 cells showing an increase in
[Ca2+]i on exposure to BeF;2 (2
nM). In these studies, 75–85% of cells showed a positive response.
The increase occurred as a single spike, multiple spikes, or as a
sustained response. (B) Effect of increasing concentrations of
BeF2 on changes in
[Ca2+]i in Fura-2/AM-loaded
macrophages. The values are mean ± SE of changes in
individual cells [Ca2+]i at 2 min
after addition of BeF2 from two experiments each using
25–30 cells. The arrow in (A) indicates the time of addition of
BeF2.
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 View larger version (28K): [in a new window] |
Figure 3. Modulations of BeF2-induced increase in
[Ca2+]i in Fura-2/AM-loaded
macrophages. (A) Oscillatory increases in
[Ca2+]i in macrophages exposed to
BeF2 (2 nM). (B) Effect of PI-PLC inhibitor U73122 (4
µM/10 min) on BeF2-induced increase in
[Ca2+]i. (C) Effect of L-type
Ca2+ channel-blocker nifedipine (5 µM) on
BeF2-induced increase in
[Ca2+]i. (D) Effect of T-type
Ca2+ channel-blocker flunarizine
• HCl (5 µM) on BeF2-induced increase in
[Ca2+]i. The inhibitors were
present during Ca2+ measurements. The data
shown in each case are representative of two to three individual
experiments, each using 20–25 cells.
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 View larger version (18K): [in a new window] |
Figure 4. Modulations of BeF2-induced increase in macrophage
cell number and [3H]thymidine incorporation. (A) Effect
of MAPK inhibitors and [Ca2+]i
chelators on BeF2-induced increase in macrophage cell
number. The respective inhibitors were added to the cultures, and the
cells were incubated for the indicated time before adding
BeF2, which was present during the overnight incubation.
The bars are: (1) buffer, (2) BeF2 (2 nM), (3) U0126 (1
µM/10 min) + BeF2, (4) PD98059 (50 µM/90 min) + BeF2, (5) SB203580 (15 µM/30 min) +
BeF2, (6) manumycin A (10 µM/60 min) +
BeF2, and (7) BAPTA/AM (10 µM/30 min) +
BeF2. The data presented are from two experiments performed
in quadruplicate and are expressed as mean ± SE. (B)
Effect of Ca2+ chelators, PKC, tyrosine
kinases, and MAPK inhibitors on BeF2-induced increase in
[3H]thymidine incorporation into macrophages. The
respective inhibitors were added to monolayers, and the cells were
incubated for the indicated time before adding BeF2, which
was present during the overnight incubation. The bars are: (1) buffer,
(2) BeF2 (2 nM), (3) BAPTA/AM (10 µM/30 min) +
BeF2, (4) chelerythrin (200 nM/10 min) +
BeF2, (5) genestein (20 µM/16 h) + BeF2,
(6) PD98059 (50 µM/90 min) + BeF2, and (7) SB 203580
(15 µM/30 min) + BeF2. The results are the mean ± SE from two to three experiments performed in
quadruplicate. (C) Effect of inhibition of IP3 generation,
L-type Ca2+ channels, ERK1, and NF-B
activation on BeF2-induced increase in
[3H]thymidine incorporation. The bars are: (1) buffer,
(2) BeF2 (2 nM), (3) U73122 (4 µM/10 min) +
BeF2, (4) BAY 11-7082 (25 µM/10 min) +
BeF2, (5) PGA1 (25 µM/10 min) +
BeF2, (6) nifedipine (5 µM/10 min) +
BeF2, and (7) U0126 (1 µM/10 min) +
BeF2. The results are mean ± SE from two
experiments performed in quadruplicate and are expressed as
[3H]thymidine incorporated/mg cellular protein.
[3H]Thymidine incorporation into cells treated with
various inhibitors used above in the absence of BeF2 was
comparable with or only slightly lower than the buffer-treated cells.
The data are significantly different at the 5% level compared with
vehicle-treated controls. (*P < 0.05).
|
The next series of studies directly examined the effects of BeF2 on the phosphorylation of several of the components of the p21ras-dependent MAPK pathway (Fig. 5 ). Specifically, MEK1 (Fig. 5A and 5B) , ERK1 (Fig. 5C and 5D) , p38 MAPK (Fig. 5E and 5F) , and JNK (Fig. 5G and 5H) phosphorylation was examined. In each case, BeF2-exposed macrophages demonstrated an approximate twofold increase in phosphorylation of these proteins. Pretreatment with BAPTA/AM abolished the action of BeF2 on up-regulating protein phosphorylation.
 View larger version (35K): [in a new window] |
Figure 5. Activation of MAPKs in macrophages by BeF2 and the role of
[Ca2+]i. The activation of MEK1
(A and B), ERK1 (C and D), p38 MAPK (E and F), and JNK (G and H) is
presented as immunoblots (A, C, E, and G), and their quantitative
distribution is as measured by PhosphorimagerTM (B, D, F, and H).
Immunoblots are representative of at least three separate experiments.
(A and B) Activation of MEK1. The bars are: (1) buffer, (2)
BeF2 (2 nM), and (3) BAPTA/AM + BeF2. (C
and D) Activation of ERK1. The bars are: (1) buffer, (2)
BeF2, and (3) BAPTA/AM + BeF2. (E and F)
Activation of p38 MAPK. The bars are: (1) buffer, (2) BeF2,
and (3) BAPTA/AM + BeF2. (G and H) Activation of JNK.
The bars are: (1) buffer, (2) BeF2, and (3) BAPTA/AM +
BeF2.
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B regulation in macrophagesB/IB complex is activated by phosphorylation of
IB [35
36
37
]. This targets IB for ubiquitination
and proteosomal degradation releasing NF-B [35
,
36
]. Thus, NF-B is activated and able to translate to
the nucleus where it stimulates gene transcription
[35
36
37
]. Therefore, we examined the effect of
BeFn on macrophages with respect to NF-B activation.
BeF2 addition caused about a twofold increase in NF-B
activation, as determined by quantifying Western blots of macrophage
extracts (Fig. 6A
and B). Next, we pretreated BeF2-exposed
macrophages to two specific inhibitors of IB phosphorylation, BAY
11-7082 and PGA1. Both agents also significantly decreased
the effect of BeF2 on increasing
[3H]thymidine uptake (Fig. 4B)
.
 View larger version (35K): [in a new window] |
Figure 6. Activation of transcription factors and expression of early genes in
macrophages exposed to BeF2. The activation of NF-B (A
and B) and CREB (C and D) is presented as immunoblots (A and C) and
their quantitative distribution (B and D). The expression of early
genes c-fos (E and F) and c-myc (G and H) is
presented as immunoblots (E and G) and their quantitative distribution
(F and H). Immunoblots are representative of three separate
experiments. (A and B) Activation of NF-B. The bars are: (1) buffer
and (2) BeF2. (C and D) Activation of CREB. The bars are:
(1) buffer and (2) BeF2. (E and F) Induction of
c-fos expression. The bars are: (1) buffer, (2)
BeF2, and (3) BAPTA/AM + BeF2. (G and H)
Induction of c-myc expression. The bars are: (1) buffer, (2)
BeF2, and (3) BAPTA/AM + BeF2.
|
BeFn exposure and regulation of c-fos
and c-myc proteins in macrophages
When quiescent cells enter the cell cycle, expression of early
response genes is necessary for progression through G1 and subsequent
proliferation [44
]. Many of these are proto-oncogenes
that encode nuclear transcription factors, which determine subsequent
expression of other genes [45
]. Expression of
c-fos is part of a mitogenic response that is required for
cell proliferation [45
]. Transcription of the
c-fos gene is regulated in part by cAMP-response element and
serum-response element [42
, 43
]. Increased
[Ca2+]i can activate
c-fos transcription through the latter mechanism, and a rise
in nuclear Ca2+ can activate c-fos
transcription through CREB phosphorylation [42
,
43
]. c-myc is effective in transcriptional
activation and cell-cycle progression [46
]. In view of
the effects of BeF2 treatment of macrophages described
above, we examined the effects of this agent on the expression of
c-fos and c-myc genes. Exposure of peritoneal
macrophages to BeF2 (2 nM) caused a 1.5- to twofold
increase in the expression of c-fos and c-myc
proteins (Fig. 6A
6B
6C
6D)
. Ca2+ chelation by
BAPTA/AM abolished this effect.
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-GDP-BeFn
complex [14
15
16
17
18
]. The effects demonstrated here contrast
to those seen in BeCl2-exposed macrophages
[27
]. This agent can also cause an increase in
[Ca2+]i in macrophages; however,
the effect is exclusively the result of entry of extracellular
Ca2+ into the cells [27
].
BeCl2-exposed macrophages show no increase in
IP3 levels at concentrations equal to or far higher than
the concentrations of BeF2 studied in the current analysis
[27
]. Moreover, BeCl2 treatment does not in
itself increase PKC activity [27
]. The data suggest that
by activating G proteins, BeFn trigger a series of events
not unlike ligands, which via receptor-binding, activate G proteins
[14
15
16
17
18
]. Subsequently, phosphoinositide degradation
produces IP3 and DAG [25
, 26
].
The former mobilizes Ca2+ from the ER via
direct interactions with its receptors, which contain a
Ca2+ channel [25
,
26
]. DAG activates PKC in the presence of
Ca2+ [25
, 26
]. The
data further indicate that these events activate
p21ras-dependent MAPK pathways and promote translocation of
critical signaling components to the nucleus, which in turn, promote
early gene activation in macrophages, thus accounting for the
proproliferative effect of BeFn species on macrophages.
These effects occur at very low concentrations of BeFn. The
majority of the effects shown here was seen at concentrations of only 2
nM BeF2. This contrasts to much higher levels of
AlFn (µM) required to affect IP3 synthesis
and subsequent effects. At higher concentrations, however,
BeF2 exerts its cytotoxic and genotoxic effects. The
mechanism(s) of BeF2 cytotoxicity and genotoxicity may be
many but may include its direct binding to the macromolecules as well
as modulation of signal-transduction cascades. It is unclear whether BeFn species are likely to be present in the environment. However, fluoride is widely available in the environment because it is added to water to harden tooth enamel. Given the low concentrations of BeFn, which induce the changes seen here, further studies might be warranted to determine the potential for environmental generation of BeFn species. This may be relevant particularly at this time, because increased amounts of beryllium are entering the environment as a result of extensive use in fiber optics, cellular communication network systems, and semiconductors [1 ].
Received August 29, 2001; revised August 29, 2001; accepted October 11, 2001.
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