Presented in part at the 18th Annual Meeting of the American Society for Bone and Mineral Research, Seattle, Washington, U.S.A., September 7–11, 1996 (J Bone Miner Res 11(Suppl 1): S382).
Protein Kinase C Involvement in Interleukin-6 Production by Parathyroid Hormone and Tumor Necrosis Factor-α in UMR-106 Osteoblastic Cells†
Article first published online: 18 FEB 2010
Copyright © 2000 ASBMR
Journal of Bone and Mineral Research
Volume 15, Issue 5, pages 885–893, May 2000
How to Cite
Sanders, J. L. and Stern, P. H. (2000), Protein Kinase C Involvement in Interleukin-6 Production by Parathyroid Hormone and Tumor Necrosis Factor-α in UMR-106 Osteoblastic Cells. J Bone Miner Res, 15: 885–893. doi: 10.1359/jbmr.2000.15.5.885
- Issue published online: 18 FEB 2010
- Article first published online: 18 FEB 2010
- Manuscript Accepted: 20 JAN 2000
- Manuscript Revised: 1 JUN 1999
- Manuscript Received: 29 OCT 1998
- parathyroid hormone;
- tumor necrosis factor-α;
- protein kinase C
The cytokine interleukin-6 (IL-6) is increased in bone and bone cells by several resorptive stimuli, including parathyroid hormone (PTH), IL-1β, and tumor necrosis factor-α (TNF-α). The current studies were designed to determine the contribution of the protein kinase C (PKC) signaling pathway to the effects of these three agents to increase IL-6 in UMR-106 rat osteoblastic cells. Cells were pretreated with vehicle (dimethylsulf-oxide [DMSO]) or the phorbol ester, phorbol 12,13-dibutyrate (PDB; 300 nM) for 48 h to down-regulate phorbol-sensitive PKC isozymes. Either PTH (0.1–10 nM), IL-1β (0.1–10 nM), or TNF-α (5 nM and 10 nM) was then added for 24 h in the continued presence of vehicle or PDB. PKC isozymes were visualized by Western immunoblotting and IL-6 was determined by bioassay. PDB pretreatment caused a partial down-regulation of the conventional α-PKC and βI-PKC isozymes and complete down-regulation of the novel δ-isoenzyme and ϵ-isozymes but it had no effect on the atypical Ξ-PKC isozyme. PDB pretreatment reduced IL-6 responses to 5 nM and 10 nM PTH by 61% and 33%, respectively, reduced IL-6 responses to 5nM and 10 nM TNF-α by 54% and 42%, respectively, and failed to inhibit the IL-6 responses to 0.1–10 nM IL-1β. The PDB pretreatment protocol significantly enhanced PTH-stimulated cyclic adenosine monophosphate (cAMP) production. The PKC inhibitor calphostin C also decreased IL-6 responses to PTH. Thus, in this osteoblast cell line, the PKC pathway is an important component of the signaling pathway for the IL-6 production stimulated by PTH and TNF-α but not that from IL-1β. (J Bone Miner Res 2000;15:885–893)
Interleukin-6 (IL-6) is a multifunctional cytokine synthesized by several different types of cells. In the bone microenvironment, IL-6 is produced by monocytes, bone marrow–derived stromal cells, and osteoblasts.(1–5) IL-6 production in bone and bone cells is stimulated by several calcemic agents including parathyroid hormone (PTH) and the cytokines IL-1 and tumor necrosis factor α (TNF-α), implicating IL-6 as a local mediator of bone resorption.(3–5) Additional studies have shown that IL-6 is a potent stimulator of osteoclast differentiation from hematopoietic progenitor cells.(6) Moreover, IL-6 elicits bone resorption in both in vivo and in vitro models containing early osteoclast precursors.(3,5,7) Further evidence of a role for IL-6 in bone remodeling is provided by studies implicating this cytokine in pathophysiological bone loss, including that associated with estrogen deficiency, Paget's disease of bone, Gorham-Stout disease, rheumatoid arthritis, and some forms of hypercalcemia of malignancy.(8)
Because of the importance of IL-6 in bone cell differentiation and remodeling, the pathways that lead to its production in bone are of interest. IL-6 production is stimulated through several different signal transduction pathways depending on both the cell type and the agonist. In bone cell models, the protein kinase C (PKC) pathway plays a significant role in IL-6 production stimulated by platelet-derived growth factor in primary rat osteoblasts and by prostaglandin F2α, in MC3T3-E1 cells.(9,10) IL-1β and TNF-α also increase PKC in the MC3T3-E1 cells; however, the activation of PKC by these agents appears to serve an autoregulatory role for IL-6 and decreases the production of the cytokine.(11,12) The well-established effect of PTH to increase cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) appears to play a role in the PTH stimulation of IL-6 production in rodent OB cells, human bone marrow stromal cells, and SaOS-2 cells, and of IL-6 messenger RNA (mRNA) in mouse calvaria.(13–16)
It is well established that PTH increases PKC activity in bone and bone cells.(17–21) A potential role for PKC in resorptive processes is suggested by studies in which PTH-dependent resorption was decreased by PKC inhibitors and chronic cotreatment with phorbol esters.(22,23) Because IL-6 has been implicated in resorption it is conceivable that PKC pathways could play a role in PTH-stimulated IL-6 production. The existing evidence for a role of PKA in PTH-stimulated IL-6 production suggests that there might be cross-talk between the two signaling pathways in mediating the production of this cytokine.
The current study was designed to determine if PKC is involved in PTH-induced IL-6 production in UMR-106 rat osteoblastic osteosarcoma cells, and if so, the possible role of cAMP in this effect. This UMR-106 cell line was selected because it has been studied extensively and it is a well-established and widely used osteoblast model in which effects of PTH have been characterized extensively.(24) We have recently characterized the PKC isozyme profile and the responses of the individual isozymes to chronic phorbol ester treatment in these cells.(25) In addition to PTH, we examined the contribution of PKC to effects of IL-1β and TNF-α on IL-6 production in the UMR-106 cells.
For the present studies, UMR-106 cells were pretreated with vehicle (dimethylsulfoxide [DMSO]) or the phorbol ester phorbol 12,13-dibutyrate (PDB) for 48 h to down-regulate phorbol-sensitive PKC isozymes. After this pretreatment, agonists were added in the continued presence of vehicle or PDB. After an additional 24 h, the culture medium was removed for determination of IL-6 and the cells were harvested for Western blotting to verify down-regulation of the phorbol-sensitive PKC isozymes. The studies reveal that in the UMR-106 cells, PKC-dependent pathways contribute significantly to the IL-6 production elicited by PTH and TNF-α but not to the IL-6 production elicited by IL-1β. The PKC-dependent effect of PTH on IL-6 production did not appear to be secondary to PTH-stimulated increases in cAMP.
MATERIALS AND METHODS
UMR-106 rat osteoblastic osteosarcoma cells (American Type Culture Collection [ATCC]), Rockville, MD, U.S.A.) were grown in 75-cm2 cell culture flasks at 37°C in a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% heat-inactivated horse serum and 100 U/ml K-penicillin G. Cells were passaged every 5–7 days with medium changes every 3 days.
For experiments, UMR-106 cells were seeded in 24-well plates at a density of 105 cells/well in 1 ml of Medium A (DMEM:Ham's F12 [1:1]) supplemented with 5% fetal bovine serum (FBS) and 80 μg/ml gentamicin). Following an overnight incubation, the medium was aspirated and 1 ml of fresh Medium A containing either vehicle (DMSO) or 300 nM phorbol PDB (Sigma Chemical Co., St. Louis, MO, U.S.A.) was added to each well. The phorbol ester dose employed in these studies was selected based on its ability to substantially down-regulate the conventional and novel PKC isozymes in UMR-106 cells, as described previously.(25) After 48 h, Medium A was removed, the cells were washed twice with DMEM, and 0.5 ml of Medium B (DMEM:Ham's F12 [1:1] supplemented with 1 μl/ml insulin, transferrin, selenous acid [ITS; Collaborative Biomedical Products, Bedford, MA, U.S.A.]) containing appropriate treatments was added to each well. To ensure continued down-regulation of phorbol-sensitive PKC isozymes, treatment with DMSO or PDB was continued in Medium B, as in Medium A. In addition, agonist treatments were initiated in half of the vehicle-pretreated wells and half of the PDB-pretreated wells. Four treatment groups (6 wells/group) were therefore present in this study: (1)DMSODMSO, 2) DMSODMSO + agonist, 3)PDBPDB, and 4) PDB PDB + agonist. The agonists employed in this study were PTH (bovine PTH(1–34); Bachem California, Inc., Torrance, CA, U.S.A.), IL-1β (recombinant human IL-1β, Calbiochem, La Jolla, CA), and TNF-α (recombinant human TNF-α; Upjohn, Kalamazoo, MI, U.S.A.). Cultures were maintained in Medium B for 24 h. At the end of the treatment period, the culture medium was removed from each well and stored at −20°C until assayed for IL-6 content. In addition, the cells in each well were harvested for Western immunoblotting to verify down-regulation of the phorbol ester-sensitive PKC isozymes. The IL-6 bioas-say and Western blotting procedure are described below.
To determine whether long-term phorbol treatment affects PTH-stimulated cAMP production, UMR-106 cells were cultured according to the phorbol ester treatment protocol described above. However, when Medium B was added to the cells two modifications were implemented. First, the Medium B was supplemented with 1 μCi/ml [3H]adenine (26.9 Ci/mmol; DuPont NEN Research Products, Boston, MA, U.S.A.) to prelabel the cells for the cAMP accumulation studies. Second, PTH treatments were initiated 24 h after the change to Medium B rather than at the time of the change. This latter modification was implemented because this study required an acute PTH treatment rather than the 24-h PTH treatment used in the IL-6 studies. After 24 h in Medium B, vehicle- and PTH-stimulated [3H]cAMP accumulation was measured in control and phorbol ester-pretreated cells, as described below.
For studies with the PKC inhibitor calphostin C, UMR-106 cells were seeded in Medium A, as outlined above. After 72 h, Medium A was removed, the cells were washed twice with DMEM, and 0.5 ml of Medium B containing appropriate treatments was added to each well. The treatments employed included vehicle, calphostin C (250 nM; Calbiochem, La Jolla, CA, U.S.A.), PTH (10 nM), and calphostin C + PTH. Because the inhibitory activity of calphostin C is dependent on exposure to light, both control and calphostin C-treated UMR-106 cells were incubated under a fluorescent light source for 1 h after addition of the treatment medium.(26) After 24 h, the culture medium was removed from each well and stored at −20°C until assayed for IL-6 content.
The concentration of IL-6 in the culture medium was determined by bioassay utilizing the IL-6-dependent 7TD1 mouse hybridoma cell line (ATCC, Rockville, MD, U.S.A.).(27) Fifty-microliter aliquots of serial 2-fold dilutions of the culture medium or 2-fold dilutions of a recombinant mouse IL-6 standard (rmIL-6; Genzyme, Cambridge, MA, U.S.A.) were prepared in 96-well culture plates with RPMI 1640 containing 10% heat-inactivated FBS, 10 mM HEPES, 50 μM 2-mercaptoethanol, and 80 μg/ml gentamicin. Next, 2000 7TD1 cells were added to each well and the cultures were incubated for 4 days at 37°C in a humidified atmosphere of 5% CO2 in air. The resulting proliferation was determined using a colorimetric assay involving 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction.(28) One unit of IL-6 was defined as the reciprocal of the medium dilution giving 50% of the maximal stimulation of proliferation by rmIL-6. PDB, calphostin C, PTH, IL-1β, or TNF-α did not have a direct effect on the proliferation of the 7TD1 cells. Two studies indicated that IL-11 is not a factor in our results. First, a mouse anti-IL-6 antibody (Upstate Biotechnology, Inc., Lake Placid, NY, U.S.A.) completely blocked 7TD1 cell proliferation induced by conditioned medium from normal mouse osteoblasts (data not shown). Second, an IL-11 neutralizing monoclonal antibody (Genetics Institute, Cambridge, MA, U.S.A.) had no effect on 7TD1 proliferation induced by conditioned medium from control or PTH-treated UMR-106 cells (data not shown). Based on these findings, IL-11 is not a contributing factor to our results.
Whole cell lysates were prepared as described previously except that after addition of lysis buffer the cells were detached from the 24-well plates by shaking the plates on an orbital shaker rather than scraping each well.(25) Cells from the 6 wells that had received identical treatments were pooled to provide sufficient sample for electrophoresis. Electrophoresis and Western blotting were carried out as described previously.(25) The isozyme-specific anti-PKC antibodies employed (rabbit polyclonals; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) were raised against C-terminal isozyme peptides. Goat, anti-rabbit peroxidase-conjugated secondary antibodies were from Sigma (St. Louis, MO, U.S.A.). Immune complexes were visualized by enhanced chemiluminescence (ECL; Amersham Life Science, Arlington Heights, IL, U.S.A.) using Kodak X-OMAT AR film (Sigma).
cAMP accumulation assay
UMR-106 cells were labeled for 24 h with 1 μCi/ml [3H]adenine (DuPont, Boston, MA, U.S.A.). After labeling, the medium was aspirated, and the cells were washed twice with DMEM to remove free [3H]adenine. cAMP formation was stimulated by the addition of DMEM containing 0.1% bovine serum albumin (BSA), 30 mM rolipram (Research Biochemicals International, Natick, MA, U.S.A.), and 10 nM PTH or its vehicle (1 mM HCl). Treatments were carried out for 10 minutes at 37°C and were terminated by aspiration of the medium and subsequent addition of 1 ml of ice-cold 5% trichloroacetic acid (TCA; Sigma, St. Louis, MO, U.S.A.). Cells were incubated with TCA for 4 h at 4°C to denature cellular proteins and to extract soluble nucleotides, including the [3H]adenine nucleotides. [3H]cAMP was separated from [3H]adenosine triphosphate (ATP) using Dowex (AG50W-X4; Bio-Rad, Hercules, CA, U.S.A.) and neutral alumina (Sigma) column chromatography, as described previously.(29) [3H]cAMP was determined by liquid scintillation counting. Before loading the columns, each sample was spiked with a known amount (∼1000 cpm) of [14C]cAMP (52.3 mCi/mmol; DuPont) to account for recovery of cAMP eluted from the columns. Individual [3H]cAMP values were then normalized through recovery of [14C]cAMP.
Data presentation and statistical analysis
Values were expressed as mean ± SEM. Statistical significance was determined by analysis of variance (ANOVA) and subsequent Fisher's least significant difference (LSD) multiple-comparison test. The probability value of p < 0.05 was used to indicate statistical significance.
PTH stimulation of IL-6 production: effect of PKC down-regulation
Treatment of UMR-106 cells for 24 h with 5 nM or 10 nM PTH produced marked increases in IL-6 production (Fig. 1). There was no detectable IL-6 production in control cells (data not shown). PDB pretreatment for 48 h, followed by cotreatment with PTH reduced the IL-6 production elicited by 5 nM PTH by 61% and that elicited by 10 nM PTH by 33% (Fig. 1). The Western blots in Fig. 2 show the effect of the PDB treatment on the PKC isozymes. The conventional (α and β1) PKC isozymes were markedly, although not completely, down-regulated by the PDB pretreatment employed in this study. The β1 antibody detected two bands, one of which was undetectable in the cells treated with PDB. The novel δ-isoenzyme and ϵ-isozyme were undetectable after the PDB treatment. The atypical Ξ-PKC isozyme was not affected by this treatment. The sensitivities of these isozymes to chronic PDB treatment are consistent with those reported previously.(25) PTH treatment for 24 h generally did not affect the total amounts of the isozymes, although a small increase in PKC-α was detected when the PDB-pretreated cells were treated with PTH.
Effect of calphostin C on PTH-stimulated IL-6 production
To further confirm that PKC is involved in PTH-stimulated IL-6 production, the PKC inhibitor calphostin C was used. Calphostin C inhibits PKC by competing at the binding site for diacylglycerol (DAG)/phorbol esters.(26) As shown in Fig. 3, calphostin C inhibited PTH-stimulated IL-6 production. Control cultures produced 65 U/ml IL-6 in response to 10 nM PTH, but cultures treated with both calphostin C (250 nM) and PTH (10 nM) produced only 11 U/ml IL-6, an 83% reduction in PTH-stimulated IL-6 production compared with the cultures without calphostin C. In the absence of PTH, there was no detectable IL-6 in control or calphostin C–treated cultures.
Effect of chronic phorbol treatment on PTH-stimulated cAMP production
To verify that the reduction in PTH-stimulated IL-6 production after PDB pretreatment was the direct result of PKC down-regulation rather than an indirect effect through the cAMP pathway, an additional study was conducted. UMR-106 cells were subjected to chronic phorbol ester pretreatment (1 μM PDB), as in the IL-6 studies. cAMP accumulation was then measured in control and phorbol-pretreated cells in response to acute PTH treatment (10 nM for 10 minutes). As shown in Fig. 4, the PDB pretreatment protocol employed in these studies significantly enhanced PTH-stimulated cAMP production in the UMR-106 cells, in contrast to its effects on PTH-stimulated IL-6 production.
IL-1β and TNF-α stimulation of IL-6 production: effect of PKC down-regulation
The PDB pretreatment protocol employed for the PTH studies also was employed to determine whether PKC is involved in IL-6 production elicited by IL-1β or TNF-α. IL-1β elicited a small but significant increase in IL-6 production in the UMR-106 cells (Fig. 5). PDB pretreatment failed to inhibit the IL-6 production elicited by either 0.1 nM or 1 nM IL-1β and slightly increased IL-6 production in response to 10 nM IL-1β.
Like PTH and in contrast to the results with IL-1β, the TNF-α–stimulated IL-6 production was significantly inhibited in PDB-pretreated cells (Fig. 6). PDB pretreatment inhibited IL-6 production elicited by 5 nM and 10 nM TNF-α by 54% and 42%, respectively. As with the PTH experiments, there was no detectable IL-6 in control or PDB-pretreated UMR-106 cells in the absence of cytokine stimulation.
Western immunoblotting for the PKC isozymes (Fig. 7) showed similar effects to those found in the PTH studies. The conventional and novel PKC isozymes were down-regulated by the PDB pretreatment, but the atypical Ξ-isozyme was not. As was seen with PTH, a slight enhancement of PKC-α by the cytokine treatments was detected in the PDB-pretreated cells.
The results presented here indicate that PKC plays a role in PTH and TNF-α–stimulated IL-6 production in UMR-106 osteoblastic cells. A substantial component of the PTH-and TNF-α–stimulated IL-6 response was lost when UMR-106 cells were subjected to chronic phorbol ester treatment before and during agonist addition. The down-regulation was confirmed by Western immunoblotting of PKC isozymes. The continued phorbol ester treatment during agonist addition was important, because our previous studies have shown the reversibility of down-regulation once the phorbol ester is removed.(25) The inhibitory effect was observed at each of the stimulatory PTH and TNF-α doses employed. In contrast, chronic phorbol treatment had no effect on the IL-6 response elicited by 0.1 nM or 1 nM IL-1β, and it enhanced IL-6 production in response to 10 nM IL-1β. These results indicate that one or more phorbol ester-sensitive PKC isozymes play a role in PTH- and TNF-α–stimulated IL-6 production in UMR-106 osteoblastic cells but that these isozymes are not critical for the IL-6 production stimulated by IL-1β. Taken together, these studies also exclude the possibility that the phorbol-mediated inhibition of the IL-6 response is the result of a nonspecific reduction in cell number.
Because there also is evidence that cAMP is important for PTH-stimulated IL-6 production in certain osteoblastic cells, the role of the PKA pathway in the current observations must be considered.(13,15) Cross-talk between PTH stimulation of phospholipase C/PKC and cAMP/PKA pathways has been shown in osteoblasts.(30–35) Acute pretreatment or cotreatment (≤1 h) with a phorbol ester has been found to enhance PTH-stimulated adenylate cyclase (AC) activity and cAMP production in UMR-106 and other osteoblastic cells, whereas longer phorbol pretreatments (2–16 h) were found to decrease PTH-stimulated AC activity, PTH receptor number, or PTH binding in osteoblasts.(30–35) In view of these previously reported interactions, we examined the effects of our PDB regimen on cAMP production. In the current studies, 72-h exposure to PDB, a treatment regimen that significantly inhibited PTH-stimulated IL-6 production, increased PTH-stimulated cAMP production in UMR-106 cells. This suggests that the phorbol-induced reduction in PTH-stimulated IL-6 that we observed was a direct result of PKC down-regulation rather than being mediated through an indirect, negative effect on the cAMP pathway. Moreover, the finding that the PKC inhibitor calphostin C also inhibits PTH-stimulated IL-6 production provides additional evidence for the direct involvement of PKC in this response.
Although the current studies indicate that PKC is involved in TNF-α–stimulated IL-6 production in UMR-106 cells, it is unclear how PKC is activated by this cytokine. In this cell line, TNF-α has no effect on inositol phosphate turnover or intracellular Ca2+, the classical pathway leading to PKC activation(36) However, in other cell types TNF-α activates a phosphatidylcholine-specific phospholipase C (PC-PLC).(37) It is possible that this enzyme also is activated by TNF-α in osteoblasts and that one of its products, DAG, is able to activate PKC. In contrast to the effects we observed with TNF-α in the UMR-106 cells, where down-regulation of PKC diminished the effect, Kozawa et al. found that in MC3T3-E1 cells PKC activation decreased TNF-α–stimulated IL-6 production, and inhibition of PKC enhanced the effect.(12) The results suggest different mechanisms for mediation of TNF-α effects in the two cell lines. TNF-α activates the transcription factor nuclear factor kappa B (NF-κB).(38) NF-κB activation has been shown to be critical for the stimulation of IL-6 production by TNF-α in ROS 17/2.8 cells.(39) There is a potential connection between the involvement of NF-κB and PKC in TNF action, because it has been shown that PKC can phosphorylate Ik;B and in this way activate NF-κB.(40) TNF-α does not stimulate cAMP production in UMR-106 osteoblastic cells.(36) Therefore, unlike PTH, the cAMP/PKA pathway is not likely to contribute to TNF-α–stimulated IL-6 production in this cell type. TNF-α has been shown to trigger many other signaling cascades in addition to the cAMP and PKC pathways, however. These cascades include phospholipase A2 (PLA2), PC-PLC, mitogen-activated protein kinase (MAPK), and two distinct types of sphingomyelinase (SMase), a plasma membrane-bound neutral (nSMase) and an intracellular endo-/lysosomal acidic sphingomyelinase (aSMase).(38) InMC3T3-E1 cells, TNF induces sphingomyelin hydrolysis and sphingosine-1-phosphate stimulates the synthesis of IL-6 through a MAPK pathway.(12,41) Additional studies are necessary to determine which of these other pathways might be involved in TNF-α–stimulated IL-6 production in osteoblasts.
The precise mechanism by which PKC influences PTH-and TNF-α–stimulated IL-6 production in UMR-106 osteoblastic cells remains to be determined. PKC could increase IL-6 by enhancing mRNA stability and/or by transcriptional activation. The former mechanism is feasible because the IL-6 gene contains 3′-UnA sequences that are believed to confer instability to mRNA.(42) Greenfield et al. showed that PTH increases IL-6 mRNA and protein levels in both MC3T3-E1 murine osteoblastic cells and UMR-106 cells, and they suggest that this was the result of transcriptional activation.(13) In SaOS-2 cells transactivation of IL-6 is stimulated by PTH and forskolin but not by phorbol-12-myristate-13-acetate, suggesting that in this cell line PKC activation does not lead to increased transcription of the gene(15)
A significant stimulatory effect of PTH and TNF-α persists in PKC–down-regulated UMR-106 cells. Although this could represent a component of the IL-6 response mediated through non-PKC pathways, there also could be a contribution of PKC to the response because there was incomplete down-regulation by PDB and possibly even the increased production of some of the phorbol-sensitive isozymes, such as was seen in Figs. 2 and 7. It is conceivable that phorbol ester-insensitive PKC isozymes also could have contributed to the effect.
Although IL-1β–stimulated IL-6 production is likely to play a role in IL-1β–induced bone resorption, the signaling pathways leading to the IL-6 response are largely undefined. Two types of IL-1 receptors affect the responses to the cytokine. The type 1 receptor mediates the biological effects, whereas the type 2 receptor is a decoy receptor that sequesters IL-1.(43,44) The murine osteoblastic MC3T3-E1 cell line possesses the type 1 IL-1 receptor, but not the type 2 receptor, and IL-1β induces IL-6 through the type 1 receptor in this cell.(45) After receptor binding and activation, IL-1β can activate both the cAMP/PKA and the PKC pathways, and each has been implicated in IL-1β–stimulated IL-6 production in various types of cells. It is possible that the cAMP pathway, rather than the PKC pathway, also plays a role in IL-1β–stimulated IL-6 production in osteoblastic cells.
In the present studies, IL-1β–stimulated IL-6 production was unaffected by PKC down-regulation, at IL-1β concentrations of 0.1 nM and 1 nM. At a high IL-1β concentration (10 nM), PDB pretreatment slightly enhanced the IL-6 response. Similar to this finding, Kozawa et al. found that in MC3T3-E1 cells the IL-1-mediated IL-6 production was enhanced by PKC inhibitors or by blocking DAG production from PC with the inhibitor D609.(11) Also, in human monocytes PKC activation decreased IL-1–stimulated IL-6 production.(46) Thus, our current results in UMR-106 cells, in which the effects of 10 nM IL-1β were enhanced by PKC down-regulation, are consistent with the findings in the MC3T3-E1 cells and monocytes. In contrast, Kim et al. found that in bone marrow stromal cells, which presumably would include osteoblast precursors, the PKC inhibitors staurosporine and calphostin C inhibited IL-1–stimulated IL-6 production.(14)
Our studies with the UMR-106 cell line show that PKC is involved in PTH and TNF-α–stimulated IL-6 production. The PKC family consists of at least 11 isozymes with unique tissue and subcellular distributions. The conventional isozymes, PKC-α, PKC-βI, PKC-βII, and PKC-γ, require phosphatidylserine (PS), DAG, and calcium (Ca2+) for activation.(47) The novel isozymes, PKC-δ, PKC-ϵ, PKC-η, and PKC-θ also require PS and DAG for activation but are Ca2+-independent.(47) The atypical isozymes, PKC-Ξ and PKC-ι/λ, require PS but are both Ca2+- and DAG-independent.(48–50) The common structural features of a given class of PKCs also confer a similar sensitivity to tumor-promoting phorbol esters, metabolically stable DAG analogs.(51) Phorbol ester-sensitive PKC isozymes are activated by acute phorbol treatment but down-regulated with more prolonged exposures.(51,52) Previously, we characterized the expression and phorbol ester-induced down-regulation of PKC isozymes in osteoblasts.(25) PKC-α, PKC-βI, PKC-βII, PKC-ϵ, PKC-Ξ, and PKC-ι/λ are expressed in normal mouse osteoblasts and in several osteoblastic cell lines, including UMR-106 cells. The novel δ-isoenzyme, η-isoenzyme, and θ-isozyme are expressed in some of the osteoblasts examined, but PKC-γ is not detectable in this cell type. Although most of the existing PKC antagonists are not selective for specific isozymes, the recent development of several isozyme-selective PKC antagonists and the use of selective antisense oligonucleotides and dominant-negative mutants should make it feasible in future studies to determine the specific isozymes involved in the PKC-dependent stimulation of IL-6 production.
The authors thank the National Institutes of Health (NIH), the Chicago Chapter of the Arthritis Foundation, and the U.S. Army Medical Research and Materiel Command for support that made this work possible. We thank Dr. Gabor Tarjan for carrying out the studies with the anti–IL-11 antibody and Dr. Paula Witt-Enderby for help with the cAMP studies. This work was supported by NIH grant AR 11262 (P.H.S.), a grant from the Chicago Chapter of the Arthritis Foundation (P.H.S.), and a fellowship (DAMD-17–94-J-4466; J.L.S.) from the U.S. Army Medical Research and Materiel Command.
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