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Molecular Endocrinology, doi:10.1210/me.2005-0389
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Molecular Endocrinology 21 (5): 1148-1162
Copyright © 2007 by The Endocrine Society

Guanosine 3',5'-Cyclic Monophosphate (cGMP)/cGMP-Dependent Protein Kinase Induce Interleukin-6 Transcription in Osteoblasts

Kate E. Broderick1, Tong Zhang1, Hema Rangaswami, Ying Zeng, Xin Zhao, Gerry R. Boss and Renate B. Pilz

Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 92093

Address all correspondence and requests for reprints to: Renate B. Pilz, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0652. E-mail: rpilz{at}ucsd.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Natriuretic peptides and nitric oxide (NO) activate the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway and play an important role in bone development and adult bone homeostasis. The cytokine IL-6 regulates bone turnover and osteoclast and osteoblast differentiation. We found that C-type natriuretic peptide and the NO donor Deta-NONOate induced IL-6 mRNA expression in primary human osteoblasts, an effect mimicked by the membrane-permeable cGMP analog 8-chlorophenylthio-cGMP (8-CPT-cGMP). Similar results were obtained in rat UMR106 osteosarcoma cells, where C-type natriuretic peptide and 8-CPT-cGMP stimulated transcription of the human IL-6 promoter and increased IL-6 secretion into the medium. Cotransfection of type I PKG enhanced the cGMP effect on the IL-6 promoter, whereas small interfering RNA-mediated silencing of PKG I expression prevented the cGMP effect on IL-6 mRNA expression. Step-wise deletion of the IL-6 promoter demonstrated a cAMP response element to be critical for transcriptional effects of cGMP, and experiments with dominant interfering proteins showed that cGMP activation of the promoter required cAMP response element binding-related proteins, and, to a lesser extent, proteins of the CAAT enhancer-binding protein and activator protein-1 (Fos/Jun) families. 8-CPT-cGMP induced nuclear translocation of type I PKG and increased cAMP response element binding-related protein phosphorylation on Ser133. PKG regulation of the IL-6 promoter appeared to be of physiological significance, because inhibitors of the NO/cGMP/PKG signaling pathway largely prevented fluid shear stress-induced increases of IL-6 mRNA in UMR106 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-6 Is A MULTIFUNCTIONAL cytokine with important (patho)physiological functions in inflammation, autoimmunity, neoplasia, hematopoietic cell differentiation, and bone metabolism (1, 2, 3). The main sources of IL-6 in bone are osteoblasts and bone marrow stromal cells, but IL-6 is also secreted by monocytes, endothelial cells, and chondrocytes (2). IL-6 plays a key role in bone remodeling and is an important physiological mediator of at least some of the actions of PTH and PTHrP (4, 5). The main effect of IL-6 in bone is stimulation of osteoclast differentiation and bone resorption, but it also has antiapoptotic and prodifferentiation effects on osteoblasts (2, 6). Mice deficient in IL-6 or its receptor are protected from bone loss caused by estrogen or androgen depletion, demonstrating IL-6 importance in bone remodeling (2, 7). In humans, IL-6 overproduction is implicated in several skeletal diseases associated with alterations in bone turnover, including rheumatoid arthritis, multiple myeloma, Paget’s disease, and polyostotic fibrous dysplasia (McCune-Albright syndrome) (1, 2, 3, 8, 9).

IL-6 production in osteogenic cells is regulated at the transcriptional level by cytokines and hormones that induce bone resorption, such as IL-1, PTH, PTHrP, and vitamin D (2, 5, 10, 11, 12). In contrast, estrogens and androgens down-regulate IL-6 expression in osteoblasts (7, 11, 13). Cytokines and hormones mediate their effects on the IL-6 promoter by multiple signal transduction pathways, including cAMP/cAMP-dependent protein kinase (PKA), protein kinase C, and MAPKs (5, 10, 12). Accordingly, the IL-6 promoter is complex and transcriptional regulation involves at least four different classes of transcription factors binding to multiple cis-acting elements: cAMP response element binding protein (CREB), CAAT/enhancer-binding proteins (C/EBPs), activator protein-1 (AP-1), and nuclear factor-{kappa}B (NF-{kappa}B) (13, 14, 15, 16, 17). IL-6 appears to be induced during mechanical stress of bone, although there are conflicting results depending on the type of mechanical stimulation (18, 19).

cGMP signaling is important for bone development and homeostasis (20, 21, 22, 23, 24, 25, 26). cGMP is generated by soluble guanylate cyclases that are activated by nitric oxide (NO) and receptor-type guanylate cyclases that are activated by natriuretic peptides. Both types of enzymes are expressed in osteogenic cells, and NO and C-type natriuretic peptide (CNP) play a role in regulating growth and differentiation of osteoblasts (24, 25, 26, 27, 28, 29, 30). cGMP has three major intracellular receptors: cGMP-dependent protein kinases (PKG I{alpha}, PKG Iß, and PKG II), phosphodiesterases, and cyclic nucleotide-gated ion channels; in addition, high cGMP concentrations can cross-activate PKA (27). Both PKG I and II are expressed in osteoblasts and chondrocytes, but they differ in their distribution within developing bones (20, 27, 31).

Transcriptional regulation by the cGMP/PKG pathway has been recognized for some time, but the mechanisms involved are only partly understood (32). For example, in a variety of cultured cells and primary tissues, c-fos, junB, and egr-1 mRNA levels are induced by NO donors, natriuretic peptides, or membrane-permeable cGMP analogs, and transcriptional regulation of c-fos by cGMP has been studied in some detail (32). We have shown that NO and cGMP activation of the c-fos promoter requires PKG activity, with PKG I and II regulating the promoter via different mechanisms (31, 33, 34, 35). We now demonstrate transcriptional regulation of the IL-6 gene by cGMP and PKG I in osteoblasts, which involves primarily CREB, with C/EBP-related proteins and the AP-1 (Fos/Jun) transcription factor also contributing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NO, CNP, and cGMP Increase Osteoblast IL-6 mRNA Expression
We used semiquantitative RT-PCR to assess the effect of cGMP and cGMP-elevating agents on IL-6 mRNA levels in rat UMR106 osteosarcoma cells and human primary osteoblasts (hPOBs). In unstimulated cells, IL-6 mRNA was at or below the limit of detection, but it was easily detectable when cells were treated for 3 h with the NO donor Deta-NONOate, the natriuretic peptide CNP, or the cell-permeable cGMP analog 8-chlorophenylthio-cGMP (8-CPT-cGMP) [UMR106 cells are shown in Fig. 1AGo and hPOBs in Fig. 1BGo; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA is shown as a loading control]. The effects of the three agents were similar to those of 8-bromo-cAMP (8-Br-cAMP), a specific PKA activator and known inducer of IL-6 mRNA in osteoblasts (14) [Fig. 1Go, A and B; 8-CPT-cAMP could not be used because it activates PKA and PKG at similar concentrations (36)].


Figure 1
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  		Fig. 1. Effect of cGMP on IL-6 mRNA Expression in Osteoblasts

A, Rat UMR106 osteosarcoma cells were left untreated (lane 1) or were treated for 3 h with 10 µM Deta-NONOate (lane 2), 100 µM 8-CPT-cGMP (lane 3), 10 nM CNP (lane 4), or 1 mM 8-Br-cAMP (lane 5). Total cytoplasmic RNA was extracted, RT-PCR with IL-6 or GAPDH primers was performed, and PCR products were analyzed on a 1% agarose gel stained with ethidium bromide. B, Human primary osteoblasts were left untreated (lane 1) or were treated for 3 h with 100 µM 8-CPT-cGMP (lane 2), 10 nM CNP (lane 3), 10 µM Deta-NONOate (lane 4), or 1 mM 8-Br-cAMP (lane 5). RT-PCR was performed as described in panel A. C, UMR 106 cells were treated with either 100 µM 8-CPT-cGMP (left half of panel) or 1 mM 8-Br-cAMP (right half of panel) for the indicated times. RNA was extracted and RT-PCR was performed as described in panel A.

 
IL-6 mRNA induction by 8-CPT-cGMP could first be detected at 1 h, peaked at 2–4 h, and declined slowly between 4 and 8 h (Fig. 1CGo; only the induction during the first 4 h is shown). This was in contrast to 8-Br-cAMP, which induced IL-6 mRNA with faster, but more short-lived, kinetics (Fig. 1CGo). Concentrations as low as 10 µM 8-CPT-cGMP induced IL-6 mRNA expression, with maximal stimulation occurring at 50–100 µM of the drug.

CNP and cGMP Increase Osteoblast IL-6 Secretion
To determine whether cGMP induction of IL-6 mRNA resulted in increased protein synthesis and secretion, UMR106 cells were treated with 10 nM CNP, 100 µM 8-CPT-cGMP, or 1 mM 8-Br-cAMP for 24 h, and IL-6 protein levels in the culture supernatants were measured by ELISA. IL-6 secretion was increased 2.5- and 3.5-fold by CNP and 8-CPT-cGMP, respectively, and almost 6-fold by 8-Br-cAMP (Fig. 2Go). Similar cAMP-induced increases in IL-6 secretion occur in murine MC3T3-E1 osteoblasts and primary rat osteoblasts (9, 37). Adding actinomycin D simultaneously with cGMP completely prevented the increase in IL-6 secretion, reducing IL-6 protein levels to less than those in control untreated cells (Fig. 2Go). Similarly, actinomycin D prevented the cGMP-induced increase in IL-6 mRNA (data not shown). These data are consistent with cGMP increasing IL-6 de novo synthesis by stimulating gene transcription and/or stabilizing IL-6 mRNA.


Figure 2
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  		Fig. 2. cGMP Stimulation of Osteoblast IL-6 Secretion

UMR106 cells were transferred to fresh medium and either received no drugs (control) or were treated for 24 h with 10 nM CNP, 100 µM 8-CPT-cGMP, 100 µM 8-CPT-cGMP plus 1 nM actinomycin, or 1 mM 8-Br-cAMP. IL-6 protein in the culture medium was determined by solid-phase ELISA. The difference between control cells and CNP- or 8-CPT-cGMP-treated cells was statistically significant (*, P < 0.01 for both comparisons).

 
cGMP Activates the IL-6 Promoter via PKG I
To determine whether cGMP stimulated IL-6 transcription, we studied the effects of cGMP on a luciferase reporter gene under control of the human IL-6 promoter. The full-length promoter was from –724 to +11 relative to the transcription start [pIL6(–724)Luc]; it included two glucocorticoid-response elements, two AP-1 response elements, a cAMP response element (CRE), a CAAT element containing two potential C/EBP binding sites in tandem, and an NF-{kappa}B response element (Fig. 3AGo and Refs. 13, 17 , and 38).


Figure 3
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  		Fig. 3. cis-Acting Elements and trans-Acting Factors Required for cGMP Responsiveness of the IL-6 Promoter

A, Schematic representation of the IL-6 promoter, with numbers indicating nucleotide positions relative to the transcription start site of the human IL-6 gene (13 17 38 ). Vertical arrows indicate promoter truncations that were tested in reporter gene assays. Boxes represent major cis-acting elements, including two glucocorticoid response elements (GRE), two AP-1-response elements (5' and 3' AP1-RE), a CRE, a CAAT element (containing two potential C/EBP binding sites), and an NF-{kappa}B-response element (NF{kappa}B-RE). B, UMR106 cells were transfected with the IL-6 promoter constructs described in panel A (nucleotide positions of the 5' truncations are indicated); all cells were cotransfected with pRSV-ß Gal and 50 ng of PKG I{alpha} expression vector. Cells were either left untreated (control) or were treated with 100 µM 8-CPT-cGMP (cGMP) for 3 h. Luciferase activities were normalized to ß-galactosidase activities as described in Fig. 4AGo, and relative luciferase activity measured in untreated cells transfected with the full-length pIL6(–724)Luc construct was assigned a value of 1. Asterisks indicate significant differences (P < 0.05) between control and 8-CPT-cGMP-treated cells. C, Cells were cotransfected with pRSV-ß Gal, PKG I{alpha}, and either full-length wild-type pIL6(–724)Luc (open bars), or pIL6(–724)Luc containing mutations in the 5' AP-1 response element (diamond-patterned bars), the CRE (filled bars), the CAAT element (with both putative C/EBP binding sites mutated, left diagonal-striped bars), the NF-{kappa}B response element (right diagonal-striped bars), or the 3' AP-1 response element (stippled bars). Half of the cultures were treated with 100 µM 8-CPT-cGMP (left panel) or 1 mM 8-Br-cAMP (right panel), and data are expressed as fold stimulation by cGMP/cAMP. *, P < 0.05 for the comparison between mutant and wild type promoter. D, Cells were transfected with wild-type pIL6(–724)Luc as described for panel B and received additionally either empty vector (E.V., open bars) or expression vectors encoding dominant negative A-CREB (left panel, filled bars), A-C/EBP (left panel, left diagonal-striped bars), A-Fos (left panel, stippled bars), or I{kappa}B32A35A (right panel, right diagonal-striped bars). Cells were left untreated (control) or were treated for 3 h with 100 µM 8-CPT-cGMP or 1 mM 8-Br-cAMP as indicated. The relative luciferase activity of untreated cells transfected with empty vector was assigned a value of 1.

 
cGMP stimulated luciferase expression from pIL–724)Luc about 2-fold in UMR106 cells (Fig. 4AGo, left panel, compare first two columns). This cGMP effect appeared to be mediated by PKG I, as indicated by the following experiments. First, cGMP induction was abolished when cells were cotransfected with a catalytically inactive, dominant negative PKG construct (Fig. 4AGo, left panel, compare second set of columns). Second, cotransfection of 50 ng of an expression vector encoding wild-type PKG I{alpha} significantly increased the effect of cGMP (Fig. 4AGo, right panel, note the different scale). The 50 ng of PKG I{alpha} vector increased PKG I levels about 3-fold over that in untransfected cells, with PKG I levels in hPOBs comparable to those in untransfected UMR106 cells (Fig. 4BGo). Treating cells with CNP also stimulated IL-6 promoter activity in PKG I{alpha}-transfected cells, albeit less efficiently than 8-CPT-cGMP (Fig. 4AGo, right panel, gray bar). Third, when UMR106 cells were transfected with a –224 to +11 IL-6 promoter construct [pIL6(–224)Luc, described further below], along with increasing amounts of the PKG I{alpha} expression vector, cGMP stimulated the IL-6 promoter in proportion to the amount of PKG transfected (Fig. 4CGo). And fourth, silencing PKG I expression with small interfering RNA (siRNA) oligoribonucleotides targeted against the common C terminus of PKG I{alpha} and Iß almost completely prevented the cGMP-induced increase in IL-6 mRNA [Fig. 4DGo; siRNA oligoribonucleotides targeted against green fluorescent protein (GFP) served as a control]. Furthermore, cotransfection of an expression vector encoding the PKA-specific (kinase) inhibitor (PKI) had no effect on cGMP-induced IL-6 promoter activity but significantly inhibited cAMP-induced promoter activity (Fig. 4EGo, compare filled and open bars representing cells transfected with empty vector vs. PKI vector, respectively). The data in Fig. 4Go, D and E, indicate that cGMP did not act through PKA cross-activation.


Figure 4
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  		Fig. 4. Activation of the IL-6 Promoter by cGMP/PKG I

UMR106 cells were used in all experiments, except as indicated. A, Cells were cotransfected with pIL6(–724)Luc, a luciferase reporter gene under control of the human IL-6 promoter including nucleotides –724 to +11 relative to the transcription start site, and the control plasmid pRSV-ß Gal. Cells additionally received 50 ng of either empty vector (E.V.), catalytically inactive, "dominant negative" (dn) PKG Iß(A516), or wild-type PKG I{alpha}. Cultures were left untreated (open bars) or were treated with 100 µM 8-CPT-cGMP (filled bars) or 10 nM CNP (gray bar) for 3 h as indicated. Luciferase activity was normalized to ß-galactosidase activity, and the relative luciferase activity of untreated cells transfected with empty vector was assigned a value of 1. Note a different scale in the left and right panels. Left panel, *, P < 0.01 for the comparison between untreated and 8-CPT-cGMP-treated cells transfected with empty vector (paired one-tail t test). B, Cells were mock-transfected or were transfected with 50 ng wild-type PKG I{alpha}. Some cells were treated with 100 µM 8-CPT-cGMP for 3 h, and equal amounts of whole-cell extract protein were analyzed by SDS-PAGE/Western blotting using an antibody directed against the C terminus of PKG I, which recognizes both PKG I{alpha} and Iß. Human primary osteoblasts (hPOBs) are shown for comparison. C, Cells were cotransfected with the reporter pIL6(–224)Luc, which includes nucleotides –224 to +11 of the IL-6 promoter, pRSV-ß Gal, and increasing amounts of expression an vector encoding wild-type PKG I{alpha}; half of the cultures were treated with 100 µM CPT-cGMP for 3 h as indicated. D, Cells were transfected with siRNA oligoribonucleotides targeted against either GFP or PKG I; 48 h later some of the cells received 100 µM 8-CPT-cGMP for 3 h. RNA was extracted and RT-PCR was performed as described in the legend to Fig. 1AGo, but including primers for PKG I. E, Cells were transfected with pIL6(–224)Luc, pRSV-ß Gal, 50 ng of PKG I{alpha}, and either empty vector (open bars) or expression vector encoding the specific PKA inhibitor peptide PKI (filled bars); some cultures were treated with 100 µM 8-CPT-cGMP or 1 mM 8-Br-cAMP to activate PKG or PKA, respectively. F, UMR106 cells were cotransfected with pIL6(–224)Luc, pRSV-ß Gal, and either empty vector (E.V.) or an expression vector for PKG I{alpha}, PKG Iß, or PKG II. As indicated, cells were left untreated or were treated with 100 µM 8-CPT-cGMP, 0.3 µM A23187 calcium ionophore, or both agents. The amounts of PKG expression vectors were chosen to produce comparable levels of PKG activity in transfected cells (PKG I{alpha}, 0.39 ± 0.10; PKG Iß, 0.41 ± 0.17; and PKG II, 0.38 ± 0.15 nmol/min/mg protein, n = 3). Reporter gene activities in panels C, E, and F were normalized as described in panel A.

 
We showed previously in osteoblasts that cGMP and calcium act synergistically to stimulate transcription of the fos promoter and that this effect is mediated more efficiently by PKG II than PKG I (31, 34). UMR106 cells and hPOBs express both PKG I and II (Ref. 31 and data not shown). We found that the calcium ionophore A23187 had no significant effect on IL-6 promoter activity and did not alter cGMP induction (Fig. 4FGo; A23187 also had no effect on IL-6 mRNA expression, data not shown). Transfection of PKG I{alpha} was most effective in augmenting the effect of cGMP on the IL-6 promoter, whereas PKG Iß was less effective and PKG II had minimal effect (Fig. 4FGo; the amounts of PKG expression vectors were titrated to produce similar levels of PKG activity). The predominant role of PKG I in regulating the IL-6 promoter is consistent with the siRNA experiments described in Fig. 4DGo. Thus, cGMP transactivation of the IL-6 and fos promoters is differentially affected by calcium and PKG isoforms, suggesting different transcriptional mechanisms.

cis-Acting DNA Elements Involved in cGMP Induction of the IL-6 Promoter
To characterize the cis-acting elements involved in cGMP regulation of the IL-6 promoter, we used a series of 5' deletion constructs (Fig. 3AGo). The constructs were cotransfected with PKG I{alpha} into UMR cells, and, similar to results shown in Fig. 4AGo, 8-CPT-cGMP increased luciferase activity about 9-fold from the full-length (–724) promoter (Fig. 3BGo, open bars). Deletion to position –224, which removes the 5' AP-1 response element as well as two glucocorticoid response elements (14, 17), yielded 13-fold stimulation by cGMP (Fig. 3BGo, filled bars), suggesting removal of some negative-acting sequences. Deletion to position –158, which removes the CRE, markedly diminished cGMP responsiveness (Fig. 3BGo, left diagonal striped bars), and further removal of the CAAT element (construct –109, right diagonal striped bars) had little additional effect; both constructs showed small, but statistically significant, residual cGMP responsiveness. Deletion to position –49, which removes an NF-{kappa}B response element as well as a second 3' AP-1 response element, resulted in a construct that no longer responded to cGMP (Fig. 3BGo, bar with horizontal stripes). All constructs demonstrated low basal luciferase activities in untreated cells, as previously described (13, 16). These results indicate that, among several cis-acting elements in the IL-6 promoter, the CRE is most important for cGMP induction in UMR106 cells.

Experiments with full-length IL-6 promoter constructs containing mutations in individual DNA elements showed that a promoter construct with a mutant CRE was nearly unresponsive to cGMP (Fig. 3CGo, left panel, filled bar), confirming a requirement of the CRE for cGMP stimulation. Mutation of the 3' AP-1 response element diminished cGMP stimulation of the promoter by about 30% (Fig. 3CGo, left panel, stippled bar; P < 0.05 for the comparison between wild type and 3'AP-1 mutant promoter). However, mutation of the 5' AP-1 response element, the CAAT element, or the NF-{kappa}B response element was without significant effect (Fig. 3CGo, left panel, diamond-patterned, and left and right diagonal-striped bars, respectively). To eliminate the effects of overexpression of PKG I{alpha}, we transfected the mutant constructs into UMR106 cells without cotransfecting PKG I{alpha}; we found similar results to those just described, albeit with less overall stimulation by cGMP. The data with cGMP are in contrast to those with cAMP: mutation of all five response elements disrupted cAMP stimulation of the promoter construct, with mutation of the CRE having the greatest effect (Fig. 3CGo, right panel); these latter results are similar to those reported by others (9, 14).

Transcription Factors Involved in cGMP Induction of the IL-6 Promoter
To examine the contribution of transcription factors likely to be involved in cGMP regulation of the IL-6 promoter, we used a series of dominant negative proteins termed A-CREB, A-C/EBP, and A-Fos. These proteins contain a CREB-, C/EBP-, or Fos-specific leucine zipper domain with an acidic amphipathic extension at the N terminus; they do not bind DNA, but form stable high-affinity heterodimers with endogenous CREB/ATF1, C/EBP, and AP-1 family members and prevent them from binding to DNA (39, 40). We previously showed that these proteins specifically inhibit their respective transcription factor families in UMR106 cells (31). We found that A-CREB and A-C/EBP inhibited basal activity of the pIL6(–224)Luc reporter gene and that A-CREB completely abolished cGMP induction of the promoter, whereas A-C/EBP and A-Fos reduced cGMP-stimulated activity by about 75% and 50%, respectively (Fig. 3DGo, left panel; compare filled, striped, and stippled bars, cells transfected with A-CREB, A-C/EBP, or A-Fos, respectively, with open bars, cells transfected with empty vector). Similar results were obtained in UMR106 cells expressing only endogenous PKG I; A-CREB completely prevented the effect of cGMP on the pIL6(–224)Luc reporter, whereas A-C/EBP and A-Fos only partly reduced it (data not shown). Expression of the dominant negative proteins had no effect on luciferase activity in cells transfected with pIL6(–49)Luc and did not alter PKG expression levels. Thus, transcriptional activation of the IL-6 gene by cGMP involves transcription factors of the CREB, C/EBP, and AP-1 families, with CREB/ATF being the most important.

cAMP activation of the IL-6 promoter is mediated partially by NF-{kappa}B (9, 14), prompting us to examine whether NF-{kappa}B-related proteins contribute to cGMP stimulation of the IL-6 promoter. We used a phosphorylation-deficient I{kappa}B mutant, which acts as a "super-suppressor" of NF-{kappa}B: this mutant I{kappa}B32A35A is resistant to stimulus-induced degradation and traps NF-{kappa}B transactivators in the cytoplasm, preventing stimulus-dependent activation of NF-{kappa}B-dependent reporter genes (41). Transfection of I{kappa}B32A35A into UMR106 cells did not affect cGMP activation of the pIL6(–724)Luc reporter but did inhibit cAMP stimulation of the promoter construct by about 65% (Fig. 3DGo, right panel). Thus, in contrast to cAMP, cGMP stimulation of the IL-6 promoter in osteoblasts does not require NF-{kappa}B activation (9, 14).

Effect of cGMP on CREB Ser133 Phosphorylation
Because cGMP activation of the IL-6 promoter required binding of a CREB/ATF-1-related protein to the CRE, and transcriptional activation of CREB requires Ser133 phosphorylation, we studied the effect of cGMP on CREB phosphorylation using a phospho-Ser-specific antibody (42). In both hPOBs and UMR106 cells, 8-CPT-cGMP or CNP increased CREB Ser133 phosphorylation by 3- to 5-fold [Fig. 5AGo for hPOBs, and B for UMR106 cells, upper panels; total amounts of CREB protein are shown in the lower panels, and the results of three independent experiments are summarized in Fig. 5CGo; in serum-starved UMR106 cells, we previously could not detect cGMP-induced CREB phosphorylation using a different phospho-CREB-specific antibody that appeared to be less sensitive (31)]. Transfection of PKG I{alpha} into UMR106 cells enhanced cGMP-induced CREB phosphorylation compared with untransfected cells (Fig. 5BGo, right panel). Immunofluorescence staining of hPOBs using a CREB phospho-Ser133-specific antibody showed low staining in untreated cells, with a significant increase in nuclear fluorescence intensity in 8-CPT-cGMP-treated hPOBs (see panel A of the supplemental figure published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).


Figure 5
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  		Fig. 5. Effect of cGMP on CREB Ser133 Phosphorylation

A and B, hPOBs (A) or UMR106 osteoblasts (B) were left untreated (lane 1) or were treated with 100 µM 8-CPT-cGMP (lane 2) or 10 nM CNP (lane 3) for 3 h. Some UMR106 cells were transfected with 50 ng of PKG I{alpha} expression vector (B, right half). Equal amounts of cell lysates were analyzed by Western blotting using an antibody specific for CREB phosphorylated on Ser133 (upper panels) or an antibody that recognizes CREB irrespective of its phosphorylation state (lower panels). C, Western blots of three independent experiments from panels A and B were scanned at two different exposures, and phospho-CREB levels were normalized to total CREB levels; the ratio of phospho-CREB/CREB in untreated cells was assigned a value of 1. Con, Control untreated cells. The difference between control and cGMP- or CNP-treated cells was statistically significant (P < 0.05).

 
Effect of cGMP on Subcellular Localization of PKG I
We examined the subcellular localization of PKG I in hPOBs by immunofluorescence staining using a PKG I-specific antibody. In untreated cells, we found predominantly cytoplasmic PKG I staining, but in hPOBs treated with 8-CPT-cGMP prominent nuclear PKG I staining was apparent (see panel B of the supplemental figure). Because we previously demonstrated in neuronal and fibroblast-like cells that induction of the fos promoter is dependent on CREB phosphorylation and nuclear translocation of PKG I, these results suggest that transcriptional activation of the osteoblast IL-6 gene by cGMP is mediated by nuclear PKG I inducing CREB phosphorylation.

Having established the critical importance of the CRE and CREB in cGMP regulation of the IL-6 promoter, we addressed the roles of C/EBPß and c-Fos/AP-1, two transcription factors potentially regulated by cGMP (32, 35, 43).

Effect of cGMP on C/EBPß Transactivation of the IL-6 Promoter
The dominant negative A-C/EBP inhibited basal and cGMP-stimulated activity of pIL6(–224)Luc (Fig. 3DGo), suggesting that C/EBP-related proteins contribute to cGMP regulation of the IL-6 promoter. However, cGMP fully stimulated pIL6(–724)Luc containing mutations in the CAAT element that eliminate C/EBP binding to this element (Fig. 3CGo). Transfecting UMR106 cells with an expression vector encoding C/EBPß increased reporter gene expression from pIL6(–224)Luc about 4-fold and augmented the effect of 8-CPT-cGMP (Fig. 6AGo). cGMP-stimulation of the CAAT mutant promoter was increased by C/EBPß cotransfection to a similar extent as observed with the wild-type promoter (data not shown). These results suggest that C/EBPß may enhance the cGMP effect independently of the CAAT element at –155, possibly by binding to other sites and/or by cooperating with other transcription factors (13, 15, 44).


Figure 6
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  		Fig. 6. Effect of cGMP on C/EBPß Transactivation of the IL-6 Promoter and c-fos Expression

A, UMR106 cells were transfected with pIL6(–224)Luc, pRSV-ß Gal, and 10 ng of PKG I{alpha} expression vector as described in Fig. 4CGo; cells were cotransfected with either empty vector or 10 ng of C/EBPß expression vector as indicated. Cells were left untreated (open bars) or were treated with 100 µM 8-CPT-cGMP for 3 h (filled bars). Luciferase activity was normalized as described in Fig. 4Go. B, UMR106 cells (left panel) and hPOBs (right panel) were serum starved over-night in 0.1% FBS and were left untreated (lanes 1) or were treated with 100 µM 8-CPT-cGMP (lanes 2) or 10 nM CNP (lanes 3) for 30 min. Total cytoplasmic RNA was extracted and subjected to semiquantitative RT-PCR using primers specific for c-fos or GAPDH as indicated.

 
In both UMR106 cells and hPOBs, immunofluorescence staining with a C/EBPß-specific antibody showed exclusively nuclear staining in untreated cells, with no significant change in fluorescence intensity in 8-CPT-cGMP-treated cells (see panel C in the supplemental figure). This is similar to the constitutive nuclear localization of C/EBPß in preadipocyte fibroblasts and HeLa cells (45, 46) but is in contrast to localization of C/EBPß in PC12 pheochromocytoma and DKO colon cancer cells, where it is primarily extranuclear and cAMP/PKA induce nuclear translocation (46, 47).

Effect of cGMP on c-fos Expression
Because cGMP activation of the IL-6 promoter was partially inhibited by A-Fos, and cGMP increases c-fos mRNA in various cell types, we examined the effect of cGMP on c-fos expression in UMR106 cells and hPOBs. Semiquantitative RT-PCR demonstrated low levels of c-fos mRNA in untreated cells, which increased after a 30-min treatment with either 8-CPT-cGMP or CNP (Fig. 6BGo, left panel for UMR106 cells, and right panel for hPOBs). Immunofluorescence staining of hPOBs with a c-Fos-specific antibody showed some fluorescence in untreated cells, with 8-CPT-cGMP increasing the intensity of nuclear c-Fos staining (see panel D in the supplemental figure). The data from Fig. 3Go, C and D, suggested that AP-1 may contribute to cGMP induction of the IL-6 promoter by binding to the 3' AP-1 response element. Consistent with this interpretation, nuclear extracts from 8-CPT-cGMP-treated UMR106 cells showed a modest (~2-fold) increase in AP-1 DNA binding activity in EMSAs with an oligodeoxynucleotide probe corresponding to the consensus AP-1 recognition site in the IL-6 promoter (data not shown).

Binding of CREB, C/EBPß, and c-Fos to the IL-6 Promoter in Intact Cells
We performed chromatin immunoprecipitation (ChIP) assays to examine interactions between CREB, C/EBPß, or c-Fos with the proximal IL-6 promoter in the context of intact chromatin. UMR106 cells were cultured in the absence or presence of 8-CPT-cGMP, proteins and DNA were cross-linked in situ by formaldehyde fixation, and sheared chromatin was subjected to immunoprecipitation with either control IgG or antibodies specific for CREB, C/EBPß, or c-Fos. As shown in the upper panel of Fig. 7Go, IL-6 promoter sequences including the core promoter elements (–188 to +61 relative to the transcription start site of the rat promoter) were amplified by PCR from the immunoprecipitates obtained with anti-CREB, -C/EBPß, or -c-Fos antibodies (lanes 5–7), with little or no detectable amplification from control IgG precipitates (lane 4). Amplification of serial dilutions of input DNA (lanes 1–3) demonstrated the PCR conditions were semiquantitative. The ß-globin promoter, which is not transcribed in UMR106 cells and does not contain consensus sequences for the three transcription factors, was not amplified, confirming assay specificity (Fig. 7Go, lower panel). These results indicate that CREB, C/EBPß, and c-Fos were bound to the IL-6 promoter in unstimulated UMR106 cells. We found no significant increase in the amount of bound factors in cGMP-treated cells (Fig. 7Go, compare lanes 9–11 to lanes 5–7). However, we do not exclude subtle, i.e. less than 2-fold, changes in transcription factor binding or an increase in c-Fos binding at earlier time points (the ChIP experiments were performed at 3 h, when the effect of cGMP on IL-6 mRNA were maximal). Consistent with the above-described results, CREB is thought to be constitutively bound to target gene promoters and its degree of activation regulated by phosphorylation on Ser133 (42). We, therefore, conclude that the effect of cGMP on the IL-6 promoter is mediated primarily by activation of constitutively bound CREB (i.e. through CREB Ser133 phosphorylation), rather than by recruitment of CREB, C/EBPß, or c-Fos to the promoter.


Figure 7
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  		Fig. 7. ChIP Analysis of CREB, C/EBPß, and c-Fos Binding to the IL-6 Promoter in Intact Cells

UMR106 cells left untreated (lanes 1–7) or treated for 3 h with 100 µM 8-CPT-cGMP (lanes 8–11) were fixed in formalin, and the fragmented chromatin was immunoprecipitated with antibodies against CREB (lanes 5 and 9), C/EBPß (lanes 6 and 10), c-Fos (lanes 7 and 11), or control IgG (lanes 4 and 8) as described in Materials and Methods. The purified DNA fragments were amplified by PCR using primers flanking the IL-6 core promoter (upper panel) or the ß-globin promoter (lower panel). Serial dilutions of input DNA were analyzed in parallel (lanes 1–3); lane 12 shows a control reaction without DNA template.

 
Role of NO and cGMP in Mechanical Stress Induction of IL-6 mRNA
Several types of mechanical stress, including fluid shear, dynamic strain, and hydrostatic pressure, play major roles in bone remodeling (48, 49). Mechanical stretch increased IL-6 mRNA and protein in lung epithelial cells, human umbilical vein endothelial cells, and a human osteoblast-like cell line, but a decrease in IL-6 secretion occurred in primary rat osteoblasts exposed to pulsed ultrasound (18, 19, 50, 51). Thus, some types of mechanical stress regulate IL-6 mRNA expression, prompting us to study the effect of fluid shear stress on IL-6 expression in UMR106 cells. We found that exposure of UMR106 cells to 12 dynes/cm2 of fluid shear stress in a parallel plate flow chamber increased IL-6 mRNA levels significantly (Fig. 8Go, compare lanes 1 and 2, sham-treated and fluid shear-exposed cells, respectively). The increase in IL-6 mRNA was largely prevented when cells were pretreated with inhibitors of NO/cGMP/PKG signaling: 500 µM L-NG-nitroarginine methyl ester (L-NAME, an NO synthase inhibitor, lane 3), 10 µM 1H- (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, a specific soluble guanylate cyclase inhibitor, lane 4), or 100 µM 8-(4-chlorophenylthio)-ß-phenyl-1, N2-ethenoguanosine-3',5'-cyclic monophosphate (Rp-CPT-PET-cGMPS, a specific PKG inhibitor, lane 5). These results suggest that the effects of fluid shear stress on IL-6 expression were mediated, at least in part, by the NO/cGMP/PKG pathway. Control experiments showed that the exposure of UMR106 cells to fluid shear stress caused a 6.1 ± 2.4-fold increase in NO production; similar results have been reported by others (52). Fluid shear stress induced IL-6 mRNA to levels somewhat higher than those observed with 8-CPT-cGMP (Fig. 8Go, compare lanes 2 and 7), and the inhibitors of NO/cGMP/PKG signaling did not completely prevent the effect of mechanical stimulation; therefore, other pathways may contribute to the fluid shear stress induction of IL-6 mRNA.


Figure 8
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  		Fig. 8. Effect of Fluid Shear Stress on IL-6 mRNA Expression

UMR106 cells were subjected to 12 dynes/cm2 of fluid shear stress for 30 min as described in Materials and Methods. Some of the cells had received 100 µM 8-CPT-cGMP, 10 µM ODQ, 500 µM L-NAME, or 100 µM Rp-CPT-PET-cGMPS for 1 h before the fluid shear stress. Sham cells were treated exactly as those exposed to fluid shear, i.e. grown on glass slides that were placed upside down in a parallel plate flow chamber, but the sham cells were not subjected to the fluid shear stress. At the end of the fluid shear exposure, RNA was extracted and RT-PCR was performed as described in the legend to Fig. 1Go.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
IL-6 is important for skeletal homeostasis because it regulates bone resorption, as well as osteoblast survival and differentiation (2, 6). Both IL-6 mRNA and protein are induced by cAMP, PTH, PTHrP, and vitamin D, and we found similar levels of induction by CNP, NO, and cGMP analogs (9, 11, 12, 37). The concentrations of CNP and the NO-donor DETA-NONOate used in these studies were equivalent to concentrations that increase intracellular cGMP, stimulate proliferation, and induce differentiation-associated genes in primary osteoblasts or immortalized osteoblast-like cells (24, 25, 26, 28, 29, 30, 53).

We found that cGMP activation of PKG I increased transcription of the IL-6 gene primarily through increased Ser133 phosphorylation of constitutively bound CREB. A mutation in the IL-6 promoter CRE or expression of a dominant negative A-CREB completely abolished cGMP stimulation of the promoter, indicating an absolute requirement of CREB-related proteins binding at the CRE. The relative increase in CREB Ser133 phosphorylation observed in CNP- or cGMP-treated hPOBs and UMR106 cells was similar in magnitude to that observed in osteoblasts stimulated with cAMP, PTH, PTHrP, or estren (5, 9, 54, 55, 56). CREB Ser133 phosphorylation leads to recruitment of coactivators and may enable CREB to cooperate with other transcription factors such as C/EBPß (31, 42). AP-1- and C/EBP-related proteins appear to contribute to cGMP regulation of the IL-6 promoter, because A-Fos and A-C/EBP partly inhibited cGMP stimulation of pIL6(–224)Luc. In addition to CREB, we found c-Fos and C/EBPß associated with the IL-6 promoter in unstimulated osteoblasts, and cGMP did not induce major changes in their DNA binding at a time of maximal cGMP-induced IL-6 mRNA expression. cGMP stimulation of c-fos mRNA was modest compared with c-fos induction by cAMP, PTH, or PTHrP (Refs. 5, 9, 55 , and 56 ; and Broderick, K. E., unpublished observation), and mutation of the 3' AP-1 binding site had only a small effect on cGMP stimulation of the IL-6 promoter, suggesting that AP-1 plays only a minor role in cGMP regulation of this promoter. C/EBPß enhanced the effect of cGMP on the IL-6 promoter independently of C/EBPß binding to the major CAAT element. This was surprising but may suggest C/EBPß cooperation with other transcription factors and/or binding to other sites (13, 15, 44). cGMP/PKG can stimulate the transactivation potential of C/EBPß in osteoblasts through regulation of glycogen synthase kinase-3 activity (43). Although we found some similarities between cGMP and cAMP regulation of IL-6 transcription, there were several differences, most notably that cAMP required NF-{kappa}B activation and binding to the NF-{kappa}B response element, whereas cGMP did not.

CREB/ATF, C/EBP, and AP-1 family proteins cooperate with each other and with the osteoblast-specific transcription factor Runx2/Cbfa1 to regulate various genes important for bone growth and homeostasis, such as osteocalcin and cyclooxygenase-2 (57, 58). Because natriuretic peptides, NO donors, and cell-permeable cGMP analogs induce osteoblast differentiation markers including osteocalcin, our finding that cGMP modulates CREB, C/EBPß, and c-Fos may have implications for cGMP regulation of other osteoblast genes (24, 25, 26, 28, 29, 53).

We showed previously that NO/cGMP-mediated induction of the c-fos promoter depends on CREB phosphorylation and nuclear translocation of PKG I in baby hamster kidney cells and rat C6 glioma cells (34, 35). PKG I constructs excluded from the nucleus because of membrane-targeting or mutation in the nuclear localization signal do not activate c-fos, despite normal catalytic activity (33, 34). We observed nuclear localization of PKG I in cGMP-treated hPOBs, and nuclear PKG I has been reported in neutrophils and some neuronal and smooth muscle cells (33, 34, 35, 59, 60); however, in other cell types, PKG I appears excluded from the nucleus (61, 62). The reason for these differences may involve extranuclear PKG-interacting proteins preventing nuclear localization of the kinase (Ref. 63 ; and Casteel, D., and R. B. Pilz, manuscript in preparation). PKG I phosphorylates CREB Ser133 in vitro, and CREB phosphorylation in cGMP-treated cells requires PKG I activity and translocation to the nucleus, suggesting that PKG I may directly phosphorylate CREB Ser133 in vivo (35). In contrast, membrane-bound PKG II mediates cGMP regulation of the fos promoter without a change in subcellular localization or CREB phosphorylation (34) and promotes synergistic activation of c-fos by calcium and cGMP in UMR106 osteoblasts (31). In our present studies, calcium did not affect cGMP-induced IL-6 promoter activity, and PKG I, but not PKG II, enhanced the effect of cGMP on the IL-6 promoter. Thus, although both PKG I and II contribute to c-fos induction by cGMP in osteoblasts, only PKG I mediates the effect of cGMP on the IL-6 promoter.

Adaptive modeling of bone in response to mechanical loading is important to maintain normal bone architecture (18, 64). During exercise-induced compression and bending of bone, mechanical stimulation of osteoblasts occurs in the form of fluid shear stress, which stimulates osteoblast endothelial NO synthase activity (52, 65). Fluid shear stress stimulates NO production in osteoblasts, endothelial cells, and other cell types, leading to increased intracellular cGMP concentration (52, 66). Mechanical stimulation of osteoblasts activates multiple signal transduction pathways leading to increased proliferation, matrix synthesis, and bone turnover, and, at least in SaOS-2 human osteoblast-like cells, an increase in IL-6 mRNA levels (18, 64, 67). We found that fluid shear stress induced IL-6 mRNA expression in UMR106 cells and that this effect was mediated, at least in part, via the NO/cGMP/PKG signaling pathway.

Inflammatory cytokines such as IL-1, TNF-{alpha}, and interferon-{gamma} increase expression of inducible NO synthase in osteoblasts, leading to high NO concentrations (68). Excessive NO production appears to contribute to inflammation-mediated osteoporosis, because inducible NO synthase-deficient mice show lower bone loss compared with wild-type mice in a model of chronic inflammation (69). Both NO production and IL-6 plasma levels are elevated in a number of inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and sepsis (70, 71). Inflammatory cytokines activate the IL-6 promoter through multiple mechanisms (72), but cytokine-induced NO production may contribute to increased IL-6 levels. This view is supported by the finding that the NO donor sodium nitroprusside increases IL-6 plasma concentrations during hypotensive anesthesia (73).

Preliminary data suggest that the cGMP/PKG signaling pathway may regulate IL-6 expression not only in osteoblasts, but also in vascular smooth muscle cells and macrophages (Ref. 4 and Broderick, K., and R. B. Pilz, unpublished observation). During tissue hypoxia/reoxygenation, macrophages secrete IL-6, and the IL-6 induction is dependent on heme oxygenase-1 activity (74). Heme oxygenase produces carbon monoxide, which stimulates soluble guanylate cyclase, leading to increased cGMP synthesis; IL-6 mRNA induction in reoxygenated macrophages is prevented by pharmacological inhibitors of soluble guanylate cyclase, suggesting involvement of cGMP (74). To our knowledge, this is the first report to directly link IL-6 expression with cGMP signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
DNA Constructs, Antibodies, and Reagents
Expression vectors for PKG I{alpha}, Iß, and II, catalytically inactive PKG Iß(A516), and the control vector pRSV-ß-Gal were described previously (31, 33, 63). Expression vectors for A-CREB, A-C/EBP, and A-Fos were from C. Vinson (39, 40); expression vectors for C/EBPß (p35), the PKA-specific inhibitor PKI, and the NF-{kappa}B inhibitor I{kappa}B32A35A were from L. Sealy (75), R. A. Maurer (76), and J. Didonato (41), respectively. The following human IL-6 promoter luciferase constructs were from B. Stein and G. Haegeman: a 5' AP-1, CRE, NF-{kappa}B, and 3' AP-1 mutant (13, 17). The CAAT mutant construct was generated in the same background as the other mutants using the Quick Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The following oligonucleotides, each generating a new restriction site, were used to sequentially mutate two potential C/EBP binding sites contained in the CAAT element (altered nucleotides are underlined): 5'-ATGCTAAAAGGACGTCACACTGCAGTTTC-TTAATAAGGT-3' and 5'-TAATAAGGTAACGTTTCAGCCCCACCCGCTCTGGCC-3'. The construct containing mutations in both sites was sequenced and is referred to as "CAAT" mutant.

An anti-C-terminal PKG I antibody was from StressGen (Victoria, British Columbia, Canada), an antibody specific for CREB phosphorylated on Ser133 was from Upstate Biotechnology (Lake Placid, NY), and antibodies specific for CREB, C/EBPß, c-Fos, and SP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA). The membrane-permeable cyclic nucleotide analogs 8-CPT-cGMP, 8-Br-cAMP, and Rp-CPT-PET-cGMPS were from Biolog (Hayward, CA), CNP and actinomycin D were from Sigma (St. Louis, MO), ODQ and L-NAME were from Calbiochem (San Diego, CA), and Deta-NONOate was from Cayman Chemicals (Ann Arbor, MI).

Cell Culture and Characterization of Human Primary Osteoblasts
Rat UMR106 osteosarcoma cells from the American Tissue Culture Collection (Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and were used at less than five passages.

Human primary osteoblasts were established from trabecular bone obtained from surgical specimens of patients undergoing joint replacement for degenerative joint disease according to an institutionally approved human subjects protocol. Bone explant cultures were established in DMEM with 10% FBS; small fragments of trabecular bone were washed extensively and incubated with Clostridium histolyticum collagenase II (2 mg/ml; Sigma) before being placed into tissue culture dishes (77). Outgrowing cells were used at passages 1–3. Cultures were characterized by positive histochemical staining for alkaline phosphatase activity in more than 85% of cells; in response to 1,25-dihydroxy-vitamin D3 (10 nM), cells demonstrated a 1.8-fold increase in alkaline phosphatase activity and induction of osteocalcin mRNA, which was undetectable in untreated cells (77). Experiments were performed in DMEM supplemented with 10% FBS unless stated otherwise.

RT-PCR
RNA was extracted using the SV Total RNA Isolation Kit (Promega, Madison, WI); 2 µg of total RNA were subjected to reverse transcription using Superscript reverse transcriptase (Invitrogen, Carlsbad, CA) and PCR was performed on 10 or 20% of the cDNA product as described (63). The PCR primers and annealing temperatures for amplification of rat and human IL-6, GAPDH, c-fos mRNA, and PKG are described in supplemental Table I, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org; PCR conditions were 1 min denaturation at 95 C, 1 min annealing at the indicated temperature, and 1 min extension at 72 C for the indicated number of cycles. Control experiments with variable amounts of input RNA demonstrated a linear increase in PCR product over a 40-fold range (0.05–2 µg).

Quantification of IL-6 by Solid-Phase ELISA
UMR106 cells were plated in 12-well dishes and grown to about 50% confluency; 24 h before harvesting, all cultures received fresh serum-containing medium, and to some cultures the indicated drugs were added. Culture supernatants were cleared by centrifugation and frozen at –20 C until analysis. Fifty-microliter samples were analyzed for IL-6 using the Quantikine M rat IL-6 ELISA kit according to the manufacturer’s instructions, with recombinant rat IL-6 serving as a standard (R&D Systems, Inc., Minneapolis, MN).

Transfections and Reporter Gene and PKG Activity Assays
UMR106 cells were transfected with Lipofectamine Plus (Invitrogen, Carlsbad, CA) as previously described (31), except that after transfection, cells were placed in serum-containing medium and treated with the indicated drugs for 3 h before harvesting. Luciferase and ß-galactosidase activities were measured as described (33). PKG activity was determined using the synthetic substrate Kemptide and the specific PKA inhibitor PKI, with cells extracted in buffer containing 1% Triton X-100 and 0.5 M NaCl to solubilize both PKG I and II (35).

Western Blot Analyses and Immunofluorescence Studies
Western blots were generated using horseradish peroxidase-coupled secondary antibodies and enhanced chemiluminescence as described (33). For immunofluorescence studies, hPOBs were grown on fibronectin-coated glass coverslips, treated with 100 µM 8-CPT-cGMP for 2 h, fixed for 10 min in 5% freshly prepared paraformaldehyde, and permeabilized in 0.3% Triton X-100. After blocking, cells were stained with antibodies specific for pCREB (1:1000), c-Fos (1:1000), C/EBPß (1:500), or PKG I (1:1000) and counterstained with the appropriate fluorescein-labeled secondary antibody. Cells were visualized using a Nikon (Melville, NY) Eclipse TE300 deconvolution microscope with Metamorph software (Molecular Devices, Sunnyvale, CA) (33); cells incubated with only secondary antibodies demonstrated no significant fluorescence signal.

Transfection of Cells with PKG siRNA
UMR106 cells were plated at 30–40% density (about 0.1 x 106 cells/well in a six-well dish) and allowed to grow overnight. The next morning, cells were transfected with 100 pmol of siRNA and 3 µl Lipofectamine 2000 (Invitrogen) in 1 ml of serum-free media for 5 h. Then, cells were placed in DMEM with 10% FBS, RNA was extracted 48 h later, and RT-PCR was performed as described above. The PKG I siRNA targeting the sequence 5'-CCGGACAUUUAAAGACAGCAA-3', and a control siRNA targeting GFP were produced by Qiagen (Valencia, CA).

EMSAs
Nuclear extracts were prepared, incubated with 5'-end-labeled oligodeoxynucleotide probes, and analyzed by nondenaturing polyacrylamide gel electrophoresis as described (31). We used oligodeoxynucleotide probes encoding an AP-1 or a SP-1 consensus site; the latter served as a loading control.

ChIP Assay
Approximately 2 x 107 UMR106 cells were incubated in situ with 1% formaldehyde for 10 min at room temperature to cross-link DNA and proteins; cells were lysed in 1 ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% Nonidet P-40, and a protease inhibitor cocktail (Calbiochem). Nuclei were spun through a cushion of 12.5% glycerol in lysis buffer and resuspended and sonicated in a chromatin precipitation buffer as described previously (31). The DNA concentration was adjusted to 0.2 µg/ml, and 100-µl aliquots were subjected to immunoprecipitation with 3 µg of control IgG or antibodies specific for CREB, C/EBPß, or c-Fos. Immunoprecipitates were washed, eluted, and heated as described previously (31). Eluates were digested with proteinase K and DNA was purified with phenol/chloroform. Semiquantitative PCR was performed with the immunoprecipitated DNA samples or serial dilutions of input template using primers flanking a sequence from –188 to +61 relative to the transcription start site of the rat IL-6 promoter [5'-CCCCCTCCTAGCTGTGATTC-3' (sense) and 5'-GAAGGGCAGATGGAGTTGAC-3' (antisense)] or a region of the ß-globin promoter (78).

Exposure of Cells to Fluid Shear Stress
UMR106 cells were plated on 75 x 38 mm glass slides at 1.5 x 106 cells/slide in DMEM containing 10% FBS. When the cells reached about 80% confluency, they were serum deprived for 24 h, and the slides were transferred to a parallel-plate flow chamber (Cytodyne Inc., San Diego, CA). Serum-free DMEM was injected into the chamber using a syringe pump at a flow rate that generated 12 dynes/cm2 of shear for 20 min. The flow chamber and accompanying apparatus were maintained at 37 C throughout the experiment. Cells grown under identical conditions, mounted into the flow chamber, but not subjected to shear stress, served as static controls. After 20 min of shear stress, cells were maintained without flow for the indicated time before total RNA was extracted and used as a template for cDNA synthesis.

Statistical Analyses
Results shown in bar graphs represent the mean ± SD of at least three independent experiments performed in duplicate. Unless stated otherwise, differences between groups were analyzed by a one-way ANOVA with a Dunnett’s post-test comparison to the control group. Photographs of gels, blots, and slides are representative experiments that were reproduced at least three times with similar results.


   ACKNOWLEDGMENTS
 
We thank W. Bugbee for providing us with operative bone specimens, G. Firestein and D. Boyle for assistance with primary osteoblast cultures, and J. Gallagher for help with deconvolution microscopy. We are grateful to J. DiDonato, G. Haegeman, S. Lohmann, R.A. Maurer, L. Sealy, B. Stein, and C. Vinson for providing DNA constructs.


   FOOTNOTES
 
R.B.P. and K.E.B. were supported by National Institutes of Health Grant R01-AR51300-06, and T.Z. was supported by American Heart Association Fellowship Grant 0525091Y.

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2007

1 K.E.B. and T.Z. contributed equally to this work. Back

Abbreviations: AP-1, Activator protein-1; 8-Br-cAMP, 8-bromo-cAMP; C/EBP, CAAT enhancer-binding protein; ChIP, chromatin immunoprecipitation; CNP, C-type natriuretic peptide; 8-CPT-cGMP; 8-para-chlorophenylthio-cGMP; CRE, cAMP response element; CREB, CRE binding protein; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; hPOBs, human primary osteoblasts; L-NAME, L-NG-nitro-arginine methyl ester; NF-{kappa}B, nuclear factor-{kappa}B; NO, nitric oxide; ODQ, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PKI, PKA-specific (kinase) inhibitor; Rp-CPT-PET-cGMPS, 8-(4-chlorophenylthio)-ß-phenyl-1, N2-ethenoguanosine-guanosine-3',5'-cyclic monophosphate; siRNA, small interfering RNA.

Received for publication September 22, 2005. Accepted for publication February 27, 2007.


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 MATERIALS AND METHODS
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