(Received for publication, January 30, 1995; and in revised form, January 12, 1996)
From the
Parathyroid hormone and other agents that stimulate bone
resorption function, at least in part, by inducing osteoblasts to
secrete cytokines that stimulate osteoclast differentiation and
activity. We previously demonstrated that parathyroid hormone induces
expression by osteoblasts of interleukin-6 and leukemia inhibitory
factor without affecting the 16 other cytokines that were examined. We
also showed that stimulation of osteoclast activity by parathyroid
hormone is dependent on activation of the cAMP signal transduction
pathway and secretion of interleukin-6 by osteoblasts. In the current
study, we demonstrate that the rapid and transient stimulation of
interleukin-6 and leukemia inhibitory factor is inhibited by
actinomycin D and superinduced by protein synthesis inhibitors, the
classical characteristics of an immediate-early gene response.
Moreover, activation of cAMP signal transduction by parathyroid hormone
and parathyroid hormone-related protein is necessary and sufficient to
induce both interleukin-6 and leukemia inhibitory factor. In addition,
cAMP analogues as well as vasoactive intestinal peptide and
isoproterenol, two neuropeptides that stimulate bone resorption by
activating cAMP signal transduction in osteoblasts, also induce
interleukin-6 and leukemia inhibitory factor in these cells. Taken
together with our previous results, this study suggests that
interleukin-6 is crucial for stimulation of bone resorption not only by
parathyroid hormone, but also by parathyroid hormone-related protein,
vasoactive intestinal peptide, and -adrenergic agonists, like
isoproterenol.
Excessive skeletal loss is caused by a disturbance in the normal
balance between bone resorption by osteoclasts, and bone formation by
osteoblasts. Knowledge of the mechanisms that regulate these processes
is fundamental to the understanding of the pathogenesis of bone loss
that occurs in conditions such as osteoporosis. In addition to forming
bone, osteoblasts appear to mediate the effect of parathyroid hormone
(PTH) ()and other resorptive agents(1, 2) .
This concept is best supported by evidence that many agents, including
PTH, stimulate bone resorption by osteoclasts that are cultured in the
presence of osteoblasts but not by osteoclasts that are cultured alone.
In most cases, resorption is also stimulated if the osteoblasts are
replaced by conditioned media from osteoblasts activated by the
resorptive agents. It is therefore thought that the primary effect of
the resorptive agents is to stimulate osteoblasts to produce soluble
cytokines that activate the osteoclasts.
We previously examined the effect of PTH on expression by osteoblasts of mRNAs encoding 18 cytokines, and found that of these only interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) were stimulated by the hormone(3, 4) . Maximal stimulation was approximately 50-fold for IL-6 and approximately 10-fold for LIF. PTH also stimulates secretion by osteoblasts of both IL-6(3, 4, 5, 6, 7, 8, 9) and LIF (10) proteins. Lack of stimulation of IL-6 and LIF by PTH in particular cell preparations is therefore likely due to investigation of osteoblasts at different developmental stages or lack of sensitivity of the methods used. Thus, such results should not be interpreted to mean that PTH does not in general stimulate IL-6 or LIF in osteoblasts (reviewed in (3) ).
Our previous detailed time course studies showed that stimulation by PTH of IL-6 and LIF mRNA expression was both rapid and transient in MC3T3-E1 osteoblastic cells, primary rat osteoblasts, and in vivo osteoblasts(3, 11) . IL-6 and LIF mRNA levels were maximal 30-60 min after stimulation and returned to baseline within 4-6 h; a pattern that is typical for immediate-early gene responses (12, 13) . In the current study, we show that the stimulation of IL-6 and LIF by PTH is inhibited by actinomycin D and superinduced by protein synthesis inhibitors, the distinguishing characteristics of an immediate-early gene response(12, 13) .
In conjunction with evidence that exogenous IL-6 and LIF can stimulate bone resorption(6, 8, 14, 15, 16) , the realization that PTH stimulates IL-6 and LIF expression led to the suggestion that production of these cytokines might be involved in stimulation of resorption by a variety of agents, including PTH(3) . In confirmation of this hypothesis, we have recently shown that an antibody directed against the IL-6 receptor can block osteoclast activation induced by PTH(17) . The importance of IL-6 is underscored by recent findings that implicate the cytokine in the increased bone resorption that occurs during estrogen withdrawal, Paget's disease, rheumatoid arthritis, multiple myeloma, hypercalcemia of malignancy, hyperparathyroidism, and Gorham-Stout disease (reviewed in (18) ).
PTH-related protein (PTHrP), which is responsible for most cases of hypercalcemia of malignancy(19) , also induces IL-6 secretion by osteoblasts(6, 7) . PTHrP and PTH utilize the same receptor to activate both the classical adenyl cyclase pathway as well as the phospholipase C pathway(20, 21) . Adenyl cyclase produces cAMP to activate protein kinase A, while phospholipase C produces inositol 1,4,5-trisphosphate and diacylglycerol to activate calcium/calmodulin-dependent kinase and protein kinase C, respectively. In order to fully understand the mechanism of action of PTH and PTHrP, it is important to clarify which signal transduction pathways are involved in the response of interest. PTH-(3-34), a truncated form of PTH, activates phospholipase C without stimulating cAMP production and can, therefore, be employed to determine whether cAMP signal transduction is involved in a particular response(22, 23) . We (17) and others (22) have utilized this partial agonist to show that activation of the cAMP pathway in osteoblasts is required for stimulation of resorption by PTH. Other workers have reached the same conclusion using inhibitors of the cAMP pathway(24, 25) .
Because
stimulation of resorption by PTH depends on activation of the cAMP
pathway and on IL-6 production, it is important to determine whether
IL-6 production depends on cAMP signaling. Activation of either the
protein kinase A, protein kinase C, or intracellular calcium pathways
can stimulate IL-6 and LIF expression by osteoblasts (26) ()or other mesenchymal
cells(27, 28, 29, 30) . Nonetheless,
it is unknown which pathways are required for stimulation of IL-6 by
PTH and PTHrP. In this study, we employed PTH-(3-34) to
distinguish between these pathways. We show here that activation of the
cAMP signaling pathway in osteoblasts is required for stimulation of
IL-6 and LIF gene expression by PTH and PTHrP. Moreover, osteoblastic
cAMP signaling is also required for stimulation of IL-6 and LIF by two
other bone resorptive agents, namely vasoactive intestinal peptide
(VIP) and isoproterenol.
All reported data are representative of multiple experiments.
PTH, PTHrP, VIP, and their analogues were obtained from Bachem
(Torrance, CA); RNA and protein synthesis inhibitors, cAMP analogues,
and -adrenergic reagents were from Sigma. All of these agents were
screened for endotoxin contamination using the colorimetric Limulus amoebocyte lysate assay (QCL-1000, Whittaker Bioproducts,
Walkersville, MD). Endotoxin levels were <0.0008 Eu/ml for the
highest concentration of each hormone used in the experiments,
<0.015 Eu/ml for the highest concentration of 8-Br-cAMP used,
<0.0075 Eu/ml for cycloheximide, and <0.034 Eu/ml for puromycin.
Moreover, none of the stimulatory agents contained endotoxin
contamination detectable by our previously described (3, 17) functional assay of cytokine induction in
MC3T3-E1 cells (see Fig. 9, for an example of this assay).
Figure 9:
PTH-(1-34), PTHrP, 8-Br-cAMP, VIP,
and isoproterenol stimulate expression by MC3T3-E1 cells of IL-6 and
LIF mRNAs, but not tumor necrosis factor (TNF
)
mRNA. Confluent MC3T3-E1 cultures were incubated with or without
indicated stimulators for 1 h prior to extraction of RNA and RT-PCR
analysis. Agents dissolved in acetic acid (25 nM PTH-(1-34), 25 nM PTH-(3-34), 25 nM PTHrP, 10 nM VIP, or 10 nM VIP-A) are shown in
the left panel where control indicates cells treated with
acetic acid alone (final concentration = 1 µM for
all cultures). Agents dissolved in water or phosphate-buffered saline
(300 µM 8-Br-cAMP, 300 µM 8-Br-cGMP, 10
µM isoproterenol (ISO), or 10 µM propranolol (PRO)) are shown in the right panel where control indicates cells treated with water and
phosphate-buffered saline (final concentration = 0.1% water and
1% phosphate-buffered saline for all cultures). Both panels include 10
µg/ml endotoxin (LPS) as a positive
control.
UMR106-01 rat osteoblast-like osteosarcoma cells (31) were
kindly provided by Dr. N. Partridge (St. Louis University). UMR106-01
cells between passage 18 and 24 were harvested using 0.02% EDTA (Sigma)
and cultured in phenol red-free (32) minimum essential media
(Life Technologies, Inc., Gaithersburg, MD) containing non-essential
amino acids (Mediatech, Herndon, VA), 2 mML-glutamine (Mediatech, Herndon, VA), 10%
heat-inactivated fetal bovine serum (HyClone, Logan, UT), 100 units/ml
penicillin (Mediatech), and 100 µg/ml streptomycin (Mediatech). All
media, serum, and additives were from lots which contained the lowest
concentration of endotoxin available. For studies of immediate-early
gene responses, confluent UMR106-01 cultures were made quiescent by
incubation in serum-free media containing 1 mg/ml bovine serum albumin
(A8412, Sigma) for 48 h prior to addition of inhibitors, stimulators,
and vehicles. For signal transduction studies, stimulators and vehicles
were added 2 h after plating (25,000 cells/cm) UMR106-01
cells in order to mimic the conditions used in our previous studies of
signal transduction during PTH-induced osteoclast
activation(17) . Murine MC3T3-E1 osteoblastic cells (33) were kindly provided by Dr. H. Tanaka (Okayama University)
and experiments were performed 1 day after reaching confluence as
described previously(3) . In experiments using RNA or protein
synthesis inhibitors, actinomycin D (10 µg/ml), cycloheximide (10
µg/ml), or puromycin (100 µg/ml) were added immediately prior
to addition of PTH. Spent media from both cell lines were consistently
negative when assayed for mycoplasma contamination by solution
hybridization to a probe complementary to mycoplasma rRNA (Gen-Probe,
San Diego, CA).
IL-6 and LIF mRNA levels were assessed by RT-PCR as described previously(3) . Murine IL-6 primers were previously described (3) and the rat IL-6 primers were modified versions of the mouse primers based on the published rat sequence(34) . LIF primers were previously described (3) and were used for both mouse and rat, as they are based on the murine sequence (35) and have been used by other workers with UMR cells(36) . Actin mRNA levels were employed in all experiments as a constitutive control using the PCR primers described in (37) . All of the PCR primer pairs flanked at least one intron to avoid false positives due to amplification of genomic DNA. Controls without cDNA and with known positive cDNAs were used in all PCR reactions. To confirm the identity of the PCR products, we showed that fragments of appropriate sizes were produced by diagnostic restriction enzymes (HpaII, MboI, and HpaII for IL-6, LIF, and actin, respectively) as described previously(3) .
IL-6 protein was determined in supernatants harvested 24 h after addition of stimulators or vehicles. In these studies, cultures were rinsed twice with phosphate-buffered saline before adding stimulators and vehicles dissolved in the media described above except containing 0.5% fetal bovine serum. IL-6 bioactivity was assessed by measuring proliferation of the B cell hybridoma B13.29 (also known as B9) as described previously(3, 7) . Purified recombinant murine IL-6 (R& Systems, Minneapolis, MN) was used in each bioassay to establish a standard curve and serve as a positive control. The presence of IL-6 was confirmed by preincubation of the culture supernatants for 30-40 min prior to addition of the B9 cells with MP5-17D2, a neutralizing rat monoclonal anti-murine/rat IL-6 antibody obtained from Dr. J. Abrams (DNAX Research Institute)(38) .
Figure 2: Cycloheximide superinduces stimulation by PTH of IL-6 and LIF expression. Confluent MC3T3-E1 cultures were incubated with or without 100 nM PTH and/or 10 µg/ml cycloheximide (CHX) for 1, 2, or 4 h prior to extraction of RNA and RT-PCR analysis. All cultures also received a mixture of vehicles, such that all were incubated with final concentrations of 1 µM acetic acid and 1.0% ethanol.
Figure 3: Puromycin superinduces stimulation by PTH of IL-6 and LIF expression. Quiescent UMR106-01 cultures were incubated with or without 100 nM PTH and/or 100 µg/ml puromycin (PURO) for 1, 2, or 4 h prior to extraction of RNA and RT-PCR analysis. All cultures also received a mixture of vehicles, such that all were incubated with final concentrations of 1 µM acetic acid and 1.0% ethanol.
Figure 1: Actinomycin D (ActD) blocks stimulation by PTH of IL-6 and LIF expression. Confluent MC3T3-E1 cultures were incubated with or without 100 nM PTH and/or 10 µg/ml actinomycin D for 1 h prior to extraction of RNA and RT-PCR analysis. All cultures also received a mixture of vehicles, such that all were incubated with final concentrations of 1 µM acetic acid and 1.0% ethanol.
Figure 4: PTH-(3-34) is unable to mimic the ability of PTH-(1-34) and PTHrP to stimulate expression of IL-6 or LIF mRNAs. UMR106-01 cells were incubated with the indicated concentrations of the hormones for 1 h prior to extraction of RNA and RT-PCR analysis. Control cells (indicated by 0 nM) were incubated with vehicle (acetic acid, final concentration = 1 µM).
To further study the role of cAMP, we examined whether membrane-permeant analogues of cAMP effect IL-6 and LIF mRNA levels. Fig. 5shows that 30-300 µM 8-Br-cAMP dose dependently stimulates expression of IL-6 and LIF. In contrast, 300 µM 8-Br-cGMP, which was used as a negative control, has no detectable effect (Fig. 5). Expression of IL-6 and LIF is also stimulated by both mono- and dibutyrl-cAMP but not by the inactive analogue, monobutyryl cyclic deoxyadenosine monophosphate (data not shown).
Figure 5: 8-Br-cAMP, but not 8-Br-cGMP, dose dependently stimulates expression of IL-6 and LIF mRNAs. UMR106-01 cells were incubated with the indicated concentrations of the cAMP analogues for 1 h prior to extraction of RNA and RT-PCR analysis. Control cells (indicated by 0 µM) were incubated with vehicle (phosphate-buffered saline, final concentration = 1%).
To determine whether
receptor-mediated intracellular cAMP is sufficient to induce IL-6 and
LIF mRNA expression, we asked whether the cytokines are also induced by
other bone resorptive agents that also stimulate cAMP in osteoblasts,
namely VIP (43) and isoproterenol(44) . VIP dose
dependently stimulates expression of IL-6 and LIF (Fig. 6), with
detectable and maximal effects at 0.1 and 10 nM VIP,
respectively. The same concentrations of
[4Cl-D-Phe,Leu
]VIP (VIP-A),
a weak VIP agonist(45) , have lesser effects on the level of
either cytokine mRNA (Fig. 6). Although VIP is thought to
function primarily through the cAMP signal transduction
system(46) , it can also stimulate other signaling pathways in
certain cell types(47, 48, 49) , possibly
including osteoblasts(50, 51) . To avoid this
potential complication, we examined the effect of the
-adrenergic
agonist, isoproterenol, which specifically stimulates cAMP signal
transduction (52) . Isoproterenol dose dependently stimulates
expression of both IL-6 and LIF mRNAs (Fig. 7), with detectable
and maximal effects at 0.1 and 10 µM, respectively. In
contrast, the same concentrations of propranolol, a
-adrenergic
antagonist which served as a negative control, did not stimulate
expression of either cytokine (Fig. 7).
Figure 6: VIP dose dependently stimulates expression of IL-6 and LIF mRNAs, while VIP-A, a weak VIP agonist, has little effect. UMR106-01 cells were incubated with the indicated concentrations of the hormones for 1 h prior to extraction of RNA and RT-PCR analysis. Control cells (indicated by 0 nM) were incubated with vehicle (acetic acid, final concentration = 1 µM).
Figure 7: Isoproterenol (ISO), but not propranolol (PRO), dose dependently stimulates expression of IL-6 and LIF mRNAs. UMR106-01 cells were incubated with the indicated concentrations of the hormones for 1 h prior to extraction of RNA and RT-PCR analysis. Control cells (indicated by 0 µM) were incubated with vehicle (water, final concentration = 0.1%).
In order to assess whether the increases in IL-6 mRNA illustrated in Fig. 4Fig. 5Fig. 6Fig. 7are reflected in augmented IL-6 protein secretion, we employed the B9 hybridoma bioassay in conjunction with an antibody that neutralizes IL-6. We previously demonstrated that PTH-(1-34) stimulates IL-6 secretion using this method(3) . Fig. 8shows that all of the agents that stimulate expression of IL-6 mRNA (PTH-(1-34), PTHrP, 8-Br-cAMP, VIP, and isoproterenol) also induce secretion of IL-6 protein. Moreover, comparison to a standard curve constructed using purified recombinant murine IL-6 allowed estimation of the amount of IL-6 in each sample. These values were 0.14 ± 0.009 pg/ml for the control cultures, 77.2 ± 3.4 pg/ml for the PTH-treated cultures, 38.9 ± 2.4 pg/ml for the PTHrP-treated cultures, 35.0 ± 1.0 pg/ml for the 8-Br-cAMP-treated cultures, 4.2 ± 0.04 pg/ml for the VIP-treated cultures, and 3.5 ± 0.1 pg/ml for the isoproterenol-treated cultures. As expected, no effect was observed when the resorptive agents were added directly to the IL-6 bioassay (data not shown). Importantly, the IL-6 bioactivity secreted in response to these agents was reduced by >93% by an antibody that specifically neutralizes authentic IL-6 (data not shown). In contrast, an anti-IL-11 antibody (11h3/19.6.1 provided by Genetics Institute) had no detectable effect on the bioactivity in the media from cultures that had been treated with the resorptive agents (data not shown).
Figure 8: PTH-(1-34), PTHrP, 8-Br-cAMP, VIP, and isoproterenol (ISO) stimulate secretion of IL-6 by UMR106-01 cells treated 2 h after plating. IL-6 levels (bottom) were measured in the B9 bioassay using the indicated concentrations of the conditioned media. Media were harvested 24 h after addition of 25 nM PTH-(1-34), 25 nM PTHrP, 300 µM 8-Br-cAMP, 10 nM VIP, or 10 µM isoproterenol. All cultures also received a mixture of vehicles, such that all were incubated with final concentrations of 1 µM acetic acid, 0.3% phosphate-buffered saline, and 0.1% water. Data are presented as the mean of triplicate determinations ± S.E. ANOVA analysis with a Bonferroni/Dunn post-hoc test compared each treatment to the same concentration of conditioned media from control cultures. For PTH-treated cultures, 0.03% conditioned media was p < 0.05 and 0.1-10% were p < 0.0005; for PTHrP, 0.03% was not significant and 0.1-10% were p < 0.0005; for 8-Br-cAMP, 0.03% was p < 0.05 and 0.1-10% were p < 0.0005; for VIP, 0.3 and 1% were not significant, 0.03 and 0.1% were p < 0.005, and 3 and 10% were p < 0.0005; for ISO, 0.3 and 1% were not significant, 0.03 and 3% were p < 0.05, and 0.1 and 10% were p < 0.0005. Replicate dishes were incubated with identical stimulators and vehicles for 1 h prior to extraction of RNA and RT-PCR analysis (top).
The
results depicted in Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8show
results with UMR106-01 cells to provide compatibility with our previous
work demonstrating that stimulation of osteoclast activity by PTH
depends on cAMP and IL-6 (17) . The same series of stimulators
and controls were employed to determine whether the cAMP signal
transduction pathway is also essential for induction of IL-6 and LIF
mRNA in MC3T3-E1 cells (Fig. 9). As was seen in UMR106-01 cells,
all of the agents that are potent stimulators of cAMP signaling
(PTH-(1-34), PTHrP, VIP, 8-Br-cAMP, and isoproterenol) strongly
increase expression of IL-6 and LIF mRNAs, while the control compounds
(PTH-(3-34), VIP-A, 8-Br-cGMP, and propranolol) had little or no
effect on expression of IL-6 and LIF mRNAs. Fig. 9also shows
that none of these agents detectably stimulate tumor necrosis factor
mRNA. Thus, stimulation of IL-6 and LIF mRNAs by these agents
cannot be attributed to endotoxin contamination, since endotoxin
strongly stimulates expression of tumor necrosis factor
mRNA (Fig. 9). This finding confirms the results of the colorimetric
endotoxin assay described under ``Materials and Methods.''
We previously showed that PTH rapidly and transiently stimulates expression of IL-6 and LIF mRNAs(3, 11) , reminiscent of classical immediate-early responses(12, 13) . The current study demonstrates that induction of these mRNAs by PTH exhibits the other characteristics of immediate-early responses; that is, it is inhibited by actinomycin D and superinduced by protein synthesis inhibitors(12, 13) . Consistent with our conclusion that this represents an immediate-early gene response, both genes contain multiple 3`-AUUUA sequences that direct rapid degradation of the previously accumulated mRNAs(53, 54) . Moreover, the 3`-untranslated regions of both the IL-6 and LIF genes (35, 55) contain multiple copies of UUUUGUA, a recently identified motif which is responsible for induction of the immediate-early gene, JE, and is found in many other immediate-early genes(56) . Since we also show here that induction of IL-6 and LIF mRNAs by PTH is dependent on cAMP signaling, we hypothesize that the transient nature of these responses, like other examples of transient cAMP-stimulated transcription(57, 58) , is dependent on inactivation of relevant transcription factors by serine/threonine protein phosphatases.
Since stimulation of osteoclast activity by PTH depends on both cAMP signal transduction and IL-6 production(17) , it is important to evaluate whether IL-6 production depends on cAMP. In the current study, we demonstrate that cAMP signal transduction is required for stimulation by PTH of both IL-6 and LIF. Moreover, PTHrP, VIP, and isoproterenol, other bone resorptive agents that stimulate cAMP signal transduction in osteoblasts(19, 43, 44) , also induce IL-6 and LIF expression by activating cAMP signal transduction. VIP and isoproterenol also stimulate IL-6 production in anterior pituitary cells (59) and astrocytes(60, 61) . In further support of a role for cAMP, the promoter region of the IL-6 gene contains multiple regulatory elements that cooperatively mediate induction by cAMP (62) and the LIF promoter contains a consensus AP-2 site(35) , which confers cAMP inducibility to other genes(63, 64, 65) . Moreover, it has recently been reported that a cAMP response element is required for stimulation of c-fos transcription by PTH in UMR106-01 cells(66) .
Although the results presented here demonstrate that the PTH/PTHrP responses depend on cAMP, they do not exclude a role for the phospholipase C pathway. We have, however, been unable to inhibit induction of IL-6 and LIF mRNAs by submaximal concentrations of PTH (500 pM) with staurosporine (10 nM), dantrolene (10 µM), or lanthanum (100 µM), either alone or in combination (data not shown). These agents block protein kinase C activation(67) , intracellular calcium release(68) , and uptake of extracellular calcium(69, 70) , respectively. These results are consistent with the demonstration that 1-alkyl-2-methylglycerol, a specific protein kinase C inhibitor, does not block the ability of PTH to stimulate IL-6 secretion(71) . We cannot rule out the possibility that the inhibitors were not fully effective and that residual calcium or protein kinase C signal transduction may have been involved in stimulation of IL-6 and LIF by PTH. Nonetheless, the current study demonstrates that if the phospholipase C pathways are involved, they act together with cAMP.
Taken together with our previous work demonstrating that IL-6 is
involved in stimulation of osteoclast activity by PTH(17) ,
this study suggests that IL-6 is also involved in stimulation of
resorption by PTHrP, VIP, and -adrenergic agonists. All of these
agents are thought to stimulate resorption through activation of
osteoblast cAMP signal
transduction(19, 43, 44) . VIP is
particularly interesting in this regard, since high concentrations of
this molecule are found near focal bone resorption sites induced by
tooth movement(72) . Thus, in response to mechanical forces,
sympathetic nerves innervating bone and periosteum (73) may
secrete VIP, which would stimulate production by osteoblasts of factors
such as IL-6 that regulate bone resorption. It has also been proposed
that VIP may be involved in the stimulation of bone resorption observed
in reflex sympathetic dystrophy(74) . Similarity of function
between PTH and VIP likely reflect homology between the intracellular
signaling domains of the two receptors(75) . In fact, it has
recently been reported that PTH and VIP antagonists are able to inhibit
activation of each other's receptor(76) .
In summary,
this study demonstrates that induction of IL-6 and LIF by PTH is an
immediate-early gene response that depends on cAMP signal transduction.
Moreover, PTHrP, VIP, and isoproterenol also induce IL-6 and LIF
expression through cAMP signal transduction. These results suggest that
stimulation of osteoclast activity by PTHrP, VIP, and isoproterenol,
like that by PTH, is mediated by production of IL-6 and possibly LIF.
We and others have also shown that many resorptive agents that activate
signaling pathways that do not involve cAMP also stimulate production
of IL-6 by osteoblasts. These include IL-1, IL-6, LIF, tumor necrosis
factor, platelet-derived growth factor, 1,25-dihydroxyvitamin
D, transforming growth factor
, endothelin, and
lipopolysaccharide (reviewed in (18) ). IL-1, IL-6, tumor
necrosis factor, transforming growth factor
, and
lipopolysaccharide have also been shown to stimulate LIF expression by
osteoblasts(3, 4, 10, 16, 17, 36, 77) .
Thus, production of IL-6, and perhaps LIF, may be critically involved
in stimulation of resorption by a wide variety of agents in addition to
those examined in this study that activate cAMP signal transduction.
Alternatively, LIF may be involved in stimulation of bone formation by
PTH (78) since LIF increases osteoblastic differentiation (36, 79) while IL-6 has been reported to inhibit this
process(80) .