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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 22, pp. 14796 –14808, May 29, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Type II cGMP-dependent Protein Kinase Mediates Osteoblast □ S Mechanotransduction Received for publication, August 21, 2008, and in revised form, March 2, 2009 Published, JBC Papers in Press, March 11, 2009, DOI 10.1074/jbc.M806486200 ‡ ‡ ‡ ‡ § § Hema Rangaswami , Nisha Marathe , Shunhui Zhuang , Yongchang Chen , Jiunn-Chern Yeh , John A. Frangos , ‡ ‡1 Gerry R. Boss , and Renate B. Pilz From the Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 92093 and the La Jolla Bioengineering Institute, La Jolla, California 92037 Continuous bone remodeling in response to mechanical load- shear stress activates various signal transduction pathways and ing is critical for skeletal integrity, and interstitial fluid flow is an initiates an anabolic response in osteocytes and osteoblasts, important stimulus for osteoblast/osteocyte growth and differ- leading to changes in gene expression and increased cell prolif- entiation. However, the biochemical signals mediating osteo- eration and differentiation (1, 5). As part of this response, a blast anabolic responses to mechanical stimulation are incom- rapid and transient increase in intracellular calcium, nitric pletely understood. In primary human osteoblasts and murine oxide (NO), and prostaglandin E occurs, and transcription of MC3T3-E1 cells, we found that fluid shear stress induced rapid genes such as c-fos, cox-2, and igf-1/2 is induced (1, 2, 5). expression of c-fos, fra-1, fra-2, and fosB/fosB mRNAs; these The transcription factor complex AP1, composed of Fos and genes encode transcriptional regulators that maintain skeletal Jun proteins, plays an essential role in bone development and integrity. Fluid shear stress increased osteoblast nitric oxide post-natal skeletal homeostasis (6). De-regulated c-fos expres- (NO) synthesis, leading to activation of cGMP-dependent pro- sion in mice interferes with normal bone development and tein kinase (PKG). Pharmacological inhibition of the induces osteosarcomas, whereas c-Fos-deficient mice develop NO/cGMP/PKG signaling pathway blocked shear-induced osteopetrosis because of an early arrest in osteoclast differenti- expression of all four fos family genes. Induction of these genes ation (7, 8). Mice overexpressing Fra-1, Fra-2, orFosB (a splice required signaling through MEK/Erk, and Erk activation was variant of FosB) exhibit a progressive increase in bone mass NO/cGMP/PKG-dependent. Treating cells with a membrane- because of enhanced osteoblast differentiation and bone for- permeable cGMP analog partly mimicked the effects of fluid mation in the presence of normal osteoclast activity (6, 9, 10). In shear stress on Erk activity and fos family gene expression. In contrast, conditional loss of Fra-1, Fra-2, or JunB leads to defec- cells transfected with small interfering RNAs (siRNA) specific tive osteoblast differentiation and severe osteopenia (6, 11, 12). for membrane-bound PKG II, shear- and cGMP-induced Erk Mechanical loading of long bones or vertebral bodies in rodents activation and fos family gene expression was nearly abolished rapidly induces c-fos mRNA in osteoblasts/osteocytes (13–16). and could be restored by transducing cells with a virus encod- The same response occurs in cultured osteoblasts exposed to ing an siRNA-resistant form of PKG II; in contrast, siRNA-me- diverse mechanical stimuli such as fluid shear stress, stretch, diated repression of the more abundant cytosolic PKG I isoform pulsed ultrasound, or gravitational force (17–24). The mecha- was without effect. Thus, we report a novel function for PKG II nism(s) regulating mechanically induced c-fos expression are in osteoblast mechanotransduction, and we propose a model poorly understood, and inhibition of calcium influx and NO or whereby NO/cGMP/PKG II-mediated Erk activation and induc- prostaglandin synthesis appears to affect c-fos induction differ- tion of c-fos, fra-1, fra-2, and fosB/fosB play a key role in the entially, depending on the type of mechanical stimulation (14, osteoblast anabolic response to mechanical stimulation. 15, 17, 19, 21). NO synthesis inhibitors prevent new bone formation induced by mechanical stimulation (14, 25), and endothelial NO synthase (eNOS, type III NOS) appears to be the predom- Mechanical stress is a primary determinant of bone growth inant NOS isoform expressed in bone (26). eNOS-deficient and remodeling; the strength of bone increases with weight mice demonstrate reduced post-natal bone mass because of bearing and muscular activity and decreases with unloading and disuse (1, 2). Weight bearing and locomotion stimulate interstitial fluid flow through the bone canalicular system, and The abbreviations used are: NO, nitric oxide; 8-pCPT-cGMP, 8-(4-chloro- phenylthio)-cGMP; CREB, cAMP-response element-binding protein; the resultant shear stress is thought to be a major mechanism eNOS, endothelial nitric-oxide synthase; Erk, extracellular signal-regu- whereby mechanical forces stimulate bone growth (1–4). Fluid lated kinase; gapd, glyceraldehyde-3-phosphate dehydrogenase; hPOB, human primary osteoblasts; IBMX, isobutylmethylxanthine; L- NAME, N-nitro-L-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazolo[4,3- * This work was supported, in whole or in part, by National Institutes of Health a]quinoxalin-1-one; MAPK, mitogen-activated protein kinase; MEK, Grant R01-AR051300 (to R. B. P., H. R., S. Z., and J. -C. Y.) and Training Grant mitogen-activated protein kinase/extracellular signal-regulated kinase T32-HL007261 (to N. M.). kinase; PAPA-NONOate, 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1- □ S The on-line version of this article (available at http://www.jbc.org) contains propanamine; PKG, cGMP-dependent protein kinase; (R )-8-pCPT-PET- supplemental Table I and Figs. 1– 4. cGMPS, 8-(4-chlorophenylthio)--phenyl-1,N -ethenoguanosine-3,5- To whom correspondence should be addressed: Dept. of Medicine and Can- cyclic monophosphorothioate (R isomer); sGC, soluble guanylate cer Center, University of California, San Diego, La Jolla, CA 92093-0652. Tel.: cyclase; siRNA, small interfering RNA; RT, reverse transcription; GFP, 858-534-8805; Fax: 858-534-1421; E-mail: [email protected]. green fluorescent protein. 14796 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009 This is an Open Access article under the CC BY license. PKG and Shear Stress-induced Gene Expression defects in osteoblast number and maturation and impaired and an antibody against the 1 subunit of sGC were from bone formation in response to mechanical stimulation and Cayman. estrogens (27–29). Osteoblasts exhibit a biphasic response to Cell Culture and Characterization of Human Primary NO donors, with low doses promoting proliferation and differ- Osteoblasts—Murine MC3T3-E1 osteoblastic cells with high entiation, and high doses inducing apoptosis (30–32). Low differentiation potential (clone 4, hereafter referred to as doses of NO donors alleviate ovariectomy-induced bone loss in MC3T3 cells) and UMR106 rat osteosarcoma cells were from rats, and a randomized, placebo-controlled trial in healthy the American Tissue Culture Collection. MC3T3 cells were post-menopausal women demonstrated increased bone forma- grown in minimal essential medium without ascorbic acid and tion in subjects receiving the NO donor isosorbide mononitrate UMR106 cells in Dulbecco’s modified Eagle’s medium; both cell (33, 34). These data indicate an important role of NO in osteo- lines were maintained in the presence of 10% fetal bovine serum blast biology, but little is known about signaling downstream of (FBS) and used at 12 passages. Human umbilical vein endo- NO in osteoblasts (35). thelial cells were isolated and cultured as described previously One of the major intracellular targets of NO is soluble gua- (48). Human primary osteoblasts (hPOBs) were established nylate cyclase (sGC), which is activated on NO binding to the from trabecular bone obtained from surgical specimens of heme prosthetic group of the enzyme. The resultant increase in patients undergoing joint replacement for degenerative joint cGMP affects multiple target proteins, including cGMP- disease, according to an institutionally approved human sub- dependent protein kinases (PKGs), phosphodiesterases, and jects protocol as described previously (41). Primary cultures cyclic nucleotide-gated ion channels (36, 37). Type I PKG were maintained for a maximum of five passages in Dulbecco’s is cytosolic and widely expressed, with high levels in smooth modified Eagle’s medium with 10% FBS and exhibited histo- muscle cells and platelets, whereas type II PKG is membrane- chemical staining for alkaline phosphatase activity in 85% of anchored, with high levels in brain and intestinal mucosa (37). cells; in addition, cells demonstrated a 16-fold induction of Studies in PKG I- and PKG II-deficient mice have demon- osteocalcin mRNA in response to 1,25-dihydroxyvitamin D strated a multitude of functions for PKG I, but there are few (10 nM) (41, 49). established functions for PKG II (37). PKG II-deficient mice are Exposure of Cells to Fluid Shear Stress—Cells were plated on dwarfs because of a severe defect in endochondral ossification 38 75-mm NaOH-etched glass slides at 0.5–1 10 cells/ at the growth plates, secondary to impaired chondrocyte pro- slide in minimal essential medium or Dulbecco’s modified liferation and differentiation (38, 39). Both PKG I and II are Eagle’s medium containing 10% FBS. After 18 h, they were expressed in osteoblasts, but their physiological significance in serum-deprived for 24 h, and the slides were transferred to a these cells is largely unknown (38, 40, 41). parallel plate flow chamber (Cytodyne Inc., San Diego). Mini- We previously showed that NO and cGMP regulate c-fos mal essential medium with 1 mg/ml fatty acid-free bovine transcription in different cell types (40, 42–47). In this study, serum albumin (Sigma) was injected into the chamber using a we found that fluid shear -stress induces c-fos, fra-1, fra-2, and syringe pump at a flow rate that generated 12 dynes/cm of fosB/fosB mRNA expression in osteoblasts via NO/cGMP, shear for up to 20 min (50). The flow chamber, media, and and we define a new function for PKG II as a regulator of gene accompanying apparatus were maintained at 37 °C throughout expression and activator of the extracellular signal-regulated the experiment. Sham-treated cells served as “static controls”; kinases (Erk)-1/2 in mechanically stimulated osteoblasts. they were grown under identical conditions and mounted into the flow chamber but were not subjected to shear stress. After EXPERIMENTAL PROCEDURES shear stress, cells were left in the chamber without flow for the Materials—Antibodies against eNOS, Erk1/2, and -tubulin indicated time before harvesting. and phospho-specific antibodies for eNOS (Ser(P) ) and Reverse Transcription (RT)-PCR—RNA was extracted using 204 TM Erk1 (Tyr(P) , clone E-4) were from Santa Cruz Biotechnol- TriReagent (Molecular Research Center, Inc.); 1 g of total ogy; an antibody specific for dually phosphorylated Erk1 RNA was subjected to reverse transcription using Super- 202 204 TM (Thr(P) /Tyr(P) ) was from Promega. Antibodies specific script reverse transcriptase (Invitrogen) and PCR was per- for vasodilator-stimulated phosphoprotein (VASP) phospho- formed on 5 or 10% of the RT product as described (51). The rylated on Ser (clone 16C2) and against the C terminus of PCR primers for amplification of murine and human glyceral- PKG I were from Calbiochem/EMD; a PKG II-specific antibody dehyde-3-phosphate dehydrogenase (gapd), c-fos, fra-1, fra-2, was from Abgent. The calcium ionophore A23187, the intracel- fosB/fosB, pkg I, and pkg II mRNA are described in supplemen- lular calcium chelator 1,2-bis(2-aminophenoxy) ethane- tal Table 1. Semi-quantitative PCR conditions for c-fos and N,N,N,N-tetraacetic acid, the MAPK/Erk kinase (MEK) gapd were 30 s of denaturation at 95 °C, 30 s annealing at 65 °C, inhibitor U0126, and the p38 inhibitor SB203580 were from and 30 s extension at 72 °C for 23 cycles; for fra-1/2 and fosB Calbiochem/EMD. The cGMP agonist 8-(4-chlorophenylthio)- annealing and extension were 45 s at 60 °C for 25 cycles. Con- GMP (8-pCPT-cGMP) and cGMP antagonist 8-(4-chlorophe- trol experiments with variable amounts of input cDNA demon- nylthio)--phenyl-1,N -ethenoguanosine-3,5-cyclic mono- strated a linear increase in a single PCR product over a20-fold phosphorothioate, R isomer (R )-8-pCPT-PET-cGMPS) were range. Quantitative RT-PCR was performed using an MX3000 p p TM from Biolog. The NOS inhibitor N-nitro-L-arginine methyl real time PCR detection system (Stratagene) and IQ SYBR ester (L-NAME), the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3- Green Supermix (Bio-Rad). Melting curves after 40 cycles con- a]quinoxalin-1-one (ODQ), the NO donor 3-(2-hydroxy-2-ni- firmed a single PCR product for each primer pair. Standard troso-1-propylhydrazino)-1-propanamine (PAPA-NONOate), curves were generated by plotting C values versus the amount MAY 29, 2009• VOLUME 284 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 14797 PKG and Shear Stress-induced Gene Expression of input RNA, and demonstrated similar amplification efficien- cies for all primers. Relative changes in mRNA expression were Ct analyzed using the 2 method, with gapd serving as an internal reference to correct for differences in RNA extraction or reverse transcription efficiencies (52). Quantitation of NO and cGMP—Nitric oxide production was monitored based on nitrite and nitrate accumulation in the medium using a two-step colorimetric assay kit according to the manufacturer’s protocol (Active Motif), but the assay was scaled down 10-fold, and samples were read using a Nano- TM Drop spectrophotometer (NanoDrop Technologies, Inc.). Experiments were performed in phenol red-free medium, with 5 10 cells/slide in the parallel plate flow chamber described above. cGMP production was measured in cell lysates using a competitive enzyme immunoassay from Cayman according to the manufacturer’s protocol. DNA and siRNA Transfections—MC3T3 cells (1.5 10 ) were transfected with 2 g of expression vector encoding human VASP in 100 l of solution V using the Amaxa Nucleoporator program D-24. After overnight recovery in full growth medium, cell from several transfections were pooled, and equal numbers of cells were plated on glass slides FIGURE 1. Effect of fluid shear stress on osteoblast c-fos, fra-1, fra-2, and for shear experiments. fosB/fosB mRNA expression. A, serum-deprived hPOBs were incubated in a parallel plate flow chamber for the indicated times; cells were exposed to The sequence targeted by siRNA in the C terminus of PKG laminar flow for the first 20 min (lanes 2– 4) or kept under static conditions I/ was 5-CCGGACAUUUAAAGACAGCAA-3 (siRNA (lane 1, Sham). At the indicated times, which include the initial 20 min of laminar flow, total cytoplasmic mRNA was isolated, and c-fos, fra-1, fra-2, fosB/ PKG-1). The sequences targeted in PKG II were 5-CTGCTT- fosB, or gapd mRNA levels were determined by semi-quantitative RT-PCR as GGAAGTGGAATACTA-3 (siRNA PKG-2a) and 5-CCG- described under “Experimental Procedures.” PCR products were separated by GGTTTCTTGGGTAGTCAA-3 (siRNA PKG-2b). siRNA oli- nondenaturing agarose gel electrophoresis and visualized by ethidium bro- mide staining. Sham-treated cells were harvested at 30 min, but similar goribonucleotides, including a control siRNA targeting green results were obtained with sham-treated cells harvested at 120 min. B, MC3T3 fluorescent protein (GFP), were produced by Qiagen. MC3T3 cells were treated as described for hPOBs in A, but c-fos, fra-1, and fra-2 mRNA cells were plated at 1.3 10 cells per well of a 6-well dish or at levels were quantified by real time RT-PCR and normalized relative to gapd mRNA levels as described under “Experimental Procedures”; the relative 5 10 cells per glass slide and were transfected 18 h later (at mRNA levels found in static controls were assigned a value of 1. p 0.05 for 40% confluency) with 100 pmol of siRNA and 3 l of Lipo- the comparison between shear-stressed and sham-treated cells for all time TM points. fectamine 2000 (Invitrogen) in 1 ml of 10% FBS-containing media. The medium was replaced 5 h later, and 18 h later, cells were transferred to medium containing 0.1% FBS. analysis to the control group; a p value of 0.05 was considered Adenovirus Infection—Adenoviral vectors encoding either statistically significant. Results shown in bar graphs represent -galactosidase (LacZ) or rat PKG II (53) were produced using the mean S.D. of at least three independent experiments, TM the pAd/CMV/V5-DEST Gateway system (Invitrogen). unless stated otherwise. All other results are representative Cells were infected in full growth medium at an multiplicity of experiments that were reproduced at least three times. infection of 30. RESULTS Western Blot Analyses, Cell Fractionation, and PKG Activity Assay—Western blots were generated using horseradish perox- Fluid Shear Stress Induces c-fos, fra-1, fra-2, and fosB/fosB idase-coupled secondary antibodies and enhanced chemilumi- mRNA Expression in Osteoblasts—Osteoblast c-fos and fosB/ nescence as described (45). To determine PKG II activity, fosB mRNA and protein are induced rapidly by mechanical MC3T3 cells were extracted by Dounce homogenization, and loading in rat vertebrae and mouse limb bones in vivo and by nuclei and cell debris were removed by centrifugation at 300 fluid shear stress in osteoblast cell lines or primary rodent g for 10 min, and the supernatant was subjected to centrifuga- osteoblasts (13–18, 54). Genetic experiments have implicated tion at 50,000 g for 30 min to generate “cytosolic” (superna- fra-1 and fra-2 in regulating osteoblast differentiation and bone tant) and “membrane” (pellet) fractions. PKG II activity in the mass (6). We found that c-fos, fra-1, fra-2, and fosB/fosB membrane fraction was determined in the presence of 1% Tri- mRNAs were strongly induced 30 min after the onset of flow in ton-X-100 and 500 mM NaCl, as described previously (40). For both MC3T3 cells and hPOBs and remained elevated for at least Western blotting, membranes were solubilized in 1% Triton 2 h (Fig. 1A shows hPOBs, results for MC3T3 cells are shown in X-100, 60 mM -octyl glucoside. Films were scanned using supplemental Fig. 1A). The housekeeping gene glyceraldehyde- ImageJ software (nih.gov). 3-phosphate dehydrogenase (gapd) was not affected by fluid Statistical Analyses—Pairwise comparison of data groups shear in either cell type. Real time RT-PCR analysis in MC3T3 was done by two-tailed Student’s t test and comparison of mul- cells showed that c-fos mRNA increased 30–40-fold at all time tiple groups by analysis of variance, with a Dunnett’s post test points, and that fra-1 and fra-2 mRNA levels increased progres- 14798 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009 PKG and Shear Stress-induced Gene Expression 40 min after the cessation of flow, suggesting a sustained activation of NOS (supplemental Fig. 1C). To determine whether fluid shear stress increased eNOS activity in MC3T3 cells, we used an antibody specific for eNOS phosphorylated on Ser ; phosphorylation of this site by multiple kinases increases enzyme activity (59). Little Ser phosphorylation was detectable in sham-treated cells, but as early as 5 min after the onset of flow, phos- phorylation increased and remained elevated at 20 min (Fig. 2B, upper panel; the lower panel shows total eNOS expression that was not changed by fluid shear). To document presence of the full FIGURE 2. Effect of fluid shear stress on osteoblast NO and cGMP production, eNOS and VASP phospho- rylation. A, MC3T3 cells were placed in the flow chamber as described in Fig. 1; they were kept either under NO/cGMP/PKG signal transduc- static conditions (gray bars) or were exposed to laminar flow (black bars) for the indicted times. At the end of tion pathway in osteoblasts, we flow (or static incubation), cells were kept in the chamber for 3 additional min, and nitrate plus nitrite (NO ) concentrations were measured in the media collected from the chamber. Thus, NO production was measured x examined MC3T3 cells for sGC and over a 3-min interval after the cessation of flow. p 0.05 was the comparison between shear-stressed and PKG. We found that sGC protein sham-treated cells for the 5- and 20-min time points. B, cells were kept under static conditions for 20 min or was easily detectable in cytosolic were exposed to fluid shear stress for the indicated times, and cell lysates were analyzed by SDS-PAGE/Western blotting using antibodies specific for eNOS phosphorylated on Ser (upper panel) or recognizing eNOS extracts of MC3T3 cells, and that irrespective of its phosphorylation state (lower panel). C, cells were extracted by Dounce homogenization and PKG I and II were present in cytoso- fractionated by differential centrifugation; cytosolic (Cyto, lane 1) and membrane (Mem, lane 2) fractions were analyzed by SDS-PAGE/Western blotting using antibodies specific for soluble guanylate cyclase 1 subunit lic and membrane fractions of (sGC, upper panel), PKG I (middle panel), or PKG II (lower panel). D, cells were placed into the flow chamber and MC3T3 cells, respectively (Fig. 2C). incubated for 15 min in the presence of 0.5 mM IBMX; cells were then either kept under static conditions (gray Fig. 2D shows that the increased NO bars) or were exposed to fluid shear stress (black bars), both in the presence of IBMX. Cells were harvested at the indicated times, and the intracellular cGMP concentration was determined as described under “Experimental in shear-stressed MC3T3 cells dra- Procedures.” E, MC3T3 cells expressing human VASP were treated as described in B, but Western blots were matically increased the intracellular probed with antibodies specific for VASP phosphorylated on Ser (upper panel)or -tubulin (lower panel). cGMP concentration; cGMP was sively up to 50- and 10-fold, respectively, at 2 h compared with measured in cells treated with the phosphodiesterase inhibitor sham-treated control cells (Fig. 1B). Basal mRNA levels of all isobutylmethylxanthine (IBMX) to block cGMP catalysis. To four fos genes were somewhat variable in sham-treated cells, determine whether the shear-induced increase in cGMP acti- explaining the relatively large standard deviations in Fig. 1B; vated PKG in intact osteoblasts in the absence of phosphodies- this may be due to the sensitivity of the cells to even small terase inhibitors, we used a phospho-specific antibody to exam- changes in fluid flow that can occur during mounting of the ine VASP phosphorylation on Ser , a preferred PKG slides. Similar results were obtained in UMR106 rat osteosar- phosphorylation site (61). VASP Ser phosphorylation was coma cells (data not shown). undetectable in sham-treated cells but increased within 5 min Fluid Shear Stress Induces NO and cGMP Production, eNOS, of initiating fluid shear stress and reached a maximum level at and VASP Phosphorylation—Mechanical stimulation of osteo- 10–20 min (Fig. 2E shows MC3T3 cells transfected with a vec- blasts or osteocytes induces NO synthesis and increased shear tor expressing human VASP, but similar results were obtained rates correlate with increased NO production (55–58). To in hPOBs, which express endogenous VASP, as shown in sup- determine the amount of NO produced in our system, we meas- plemental Fig. 1D). ured the concentration of stable NO oxidation products Inhibition of NO/cGMP Signaling Blocks Shear-induced c-fos, (nitrites and nitrates, referred to as NO ) in media collected 3 fra-1, fra-2, and fosB/fosB mRNA Expression—The experi- min after the end of flow, allowing calculation of the rate of NO ments in Figs. 1 and 2 show that fluid shear stress leads to rapid production during this 3-min period (Fig. 2A). In MC3T3 cells NO and cGMP accumulation, PKG activation, and induction exposed to 5 min of fluid shear, NO production nearly doubled of fos family members but do not establish a causal relationship compared with sham-treated cells, and it reached 1.5 0.1 between NO/cGMP/PKG signaling and increased gene expres- nmol/min/10 cells after 20 min. In contrast, 20 min of fluid sion. To address this question, we treated MC3T3 cells and shear stress increased NO production to 3.4 0.4 nmol/min/ hPOBs for 1 h with L-NAME (to inhibit NOS), ODQ (to inhibit 10 cells in human umbilical vein endothelial cells (supplemen- sGC), or (R )-8-pCPT-PET-cGMPS (to inhibit PKG) prior to tal Fig. 1B). The rate of NO production in MC3T3 cells is fluid shear stress. We found that all three agents severely comparable with that of primary mouse osteoblasts exposed to reduced shear induction of c-fos, fra-1, fra-2, and fosB/fosB pulsating fluid flow (58). The NO concentration in the mRNA, without affecting gapd mRNA (Fig. 3A shows semi- medium of shear-stressed MC3T3 cells continued to rise up to quantitative RT-PCR results for hPOBs, and Fig. 3B summa- MAY 29, 2009• VOLUME 284 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 14799 PKG and Shear Stress-induced Gene Expression fluid shear-stressed cells to the level found in sham-treated control cells (Fig. 3C); all three agents blocked shear-induced VASP Ser phos- phorylation (Fig. 3D); and ODQ and (R )-8-pCPT-PET-cGMPS blocked VASP phosphorylation in cells treated with the NO donor PAPA- NONOate (data not shown). These results suggest that fluid shear stress induction of fos family genes requires PKG activation by NO/cGMP. cGMP Partly Mimics the Effects of Fluid Shear Stress on c-fos, fra-1, fra-2, and fosB/fosB mRNA Expression, and cGMP Cooperates with Calcium—To determine whether direct PKG activation can reproduce the effects of fluid shear stress on fos family gene expression, we treated cells with a membrane- permeable cGMP agonist. Exposure of serum-starved MC3T3 cells or hPOBs to 8-pCPT-cGMP for 30 min, 1 h, or 2 h increased c-fos, fra-1, fra-2, and fosB/fosB mRNA expression, but when cells were pre- treated with the PKG inhibitor (R )- 8-pCPT-PET-cGMPS, the effects of 8-pCPT-cGMP on all four fos family genes were abolished (Fig. 4A shows hPOBs, and Fig. 4B summarizes real time RT-PCR results for c-fos, fra-1, and fra-2 in MC3T3 cells, with black and gray bars representing cells treated with 8-pCPT-cGMP FIGURE 3. Inhibition of NO/cGMP signaling prevents shear-induced c-fos, fra-1, fra-2, and fosB/fosB in the absence and presence of the mRNA expression. A, serum-deprived hPOBs were placed in a flow chamber and were incubated for 1 h with PKG inhibitor, respectively). (R )- either culture medium alone (lanes 1 and 2) or with medium containing 4 mML-NAME (lane 3), 10 M ODQ (lane p 4), or 100 M (R )-8-pCPT-PET-cGMPS ((Rp)-cGMPS, lane 5). Cells were then either kept under static conditions 8-pCPT-PET-cGMPS prevented (lane 1) or were exposed to laminar flow for 20 min (lanes 2–5). Ten minutes after the cessation of flow, total RNA 259 VASP Ser phosphorylation in was extracted, and c-fos, fra-1, fra-2, fosB/fosB, or gapd mRNA levels were determined by semi-quantitative cells treated with 8-pCPT-cGMP, RT-PCR as described in Fig. 1A. B, MC3T3 cells were treated as described for hPOBs in A, but c-fos, fra-1, and fra-2 mRNA levels were quantified after 60 min by real time RT-PCR as described in Fig. 1B. p 0.05 for the compar- demonstrating effective inhibition ison between control cells receiving no drug and cells treated with L-NAME (L-N), ODQ, or (R )-cGMPS (Rp). of PKG activity by the cGMP antag- C, MC3T3 cells were kept under static conditions (gray bars) or were exposed to laminar flow for 20 min (black bars); 5 min after the cessation of flow, media were collected from the chamber, and nitrate plus nitrite (NO ) onist (Fig. 4C). concentrations were measured. D, MC3T3 cells expressing human VASP were incubated for 1 h with medium When MC3T3 cells were treated alone (lanes 1 and 2) or with medium containing 4 mML-NAME (lane 3), 10 M ODQ (lane 4), or 100 M (R )-8- with the NO donor sodium nitro- pCPT-PET-cGMPS (Rp-cGMPS, lane 5). Cells were kept under static conditions (lane 1) or were exposed to 20 min of fluid shear stress (lanes 2–5), and 10 min after the cessation of flow, cell lysates were analyzed by SDS-PAGE/ prusside to activate PKG through Western blotting using antibodies specific for VASP phosphorylated on Ser (upper panel)or -tubulin (lower sGC stimulation, c-fos, fra-1 and panel). fra-2 mRNAs were induced to a rizes real time PCR results for c-fos, fra-1 and fra-2 in MC3T3 similar extent as seen with 8-pCPT-cGMP (compare Fig. 4D to cells). The three inhibitors almost completely prevented shear Fig. 4B). However, we noticed that sodium nitroprusside and induction of c-fos and fra-2 mRNAs; however, there was some 8-pCPT-cGMP induced the fos genes to a lesser extent than residual shear induction of fra-1 mRNA, suggesting that some observed in fluid shear-stressed MC3T3 cells (compare the y NO/cGMP/PKG-independent mechanism may contribute to axis in Fig. 4, B and D, with that in Fig. 1B), suggesting that the the mechanical stimulation of fra-1. Control experiments dem- NO/cGMP/PKG signaling pathway cooperates with other onstrated that the drugs inhibited NO/cGMP/PKG signaling pathways to induce fos family genes in mechanically stimulated effectively; L-NAME reduced the rate of NO production in osteoblasts. 14800 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009 PKG and Shear Stress-induced Gene Expression cium influx via mechano-sensitive and/or L-type voltage-gated cal- cium channels; calcium chelation prevents fluid shear stress-induced c-fos and fosB/fosB expression (17, 19, 54, 62–65). We confirmed these latter results, and we found that treating MC3T3 cells with 1,2- bis(2-aminophenoxy) ethane-N, N,N,N-tetraacetic acid or EGTA to chelate intra- or extracellular cal- cium, respectively, also reduced fluid shear stress induction of fra-1 and -2 mRNA (supplemental Fig. 2A). We previously showed that cal- cium and cGMP cooperate to increase c-fos mRNA expression (40, 47). Therefore, we examined the effect of the calcium ionophore A23187 alone and in combination with 8-pCPT-cGMP on the expres- sion of all four fos family genes in MC3T3 cells and hPOBs. Increasing the intracellular calcium concen- tration with A23187 stimulated expression of c-fos, fra-1, fra-2, and fosB/fosB mRNA to a similar extent as treatment with 8-pCPT- cGMP; together the effect was at least additive (Fig. 4E and supple- mental Fig. 2B show results for MC3T3 cells, but similar results were obtained in hPOBs). Com- bined treatment with calcium iono- phore plus cGMP induced c-fos and fra-1/2 mRNAs to levels compara- ble with those found in shear- stressed osteoblasts (compare Fig. 4E and Fig. 1B). These results sug- gest that induction of fos family genes in shear-stressed osteoblasts is due to a combined effect of increased intracellular calcium and FIGURE 4. cGMP partly mimics the effects of fluid shear stress on osteoblast c-fos, fra-1, fra-2, and fosB/ fosB mRNA expression. A, serum-deprived hPOBs were incubated for1hinthe absence (lanes 1– 4)or cGMP, although calcium and cGMP presence of 100 M (R )-8-pCPT-PET-cGMPS (Rp-cGMPS, lanes 5 and 6) before adding 50 M 8-pCPT-cGMP signaling may be partly inter- (cGMP) to all cells for the indicated times. c-fos, fra-1, fra-2, fosB/fosB or gapd mRNA levels were determined by semi-quantitative RT-PCR as described in Fig. 1A. B, MC3T3 cells were incubated in the absence (black bars)or dependent, as discussed below. presence (gray bars)of(R )-8-pCPT-PET-cGMPS and then stimulated with 8-pCPT-cGMP as described in A for Shear- and cGMP-induced c-fos, hPOBs, but c-fos, fra-1, and fra-2 mRNA levels were quantified by real time RT-PCR. Relative mRNA levels fra-1, fra-2, and fosB/fosB mRNA (normalized to gapd) measured in mock-treated cells were assigned a value of 1. p 0.05 for the comparison between cells treated with (R )-cGMPS plus cGMP versus cGMP alone for all time points. C, hPOBs were prein- Expression Is Mediated by PKG II cubated in the absence (lanes 1 and 2) or presence (lane 3)of100M (R )-8-pCPT-PET-cGMPS for 1 h and treated and Not PKG I—Having established with 50 M 8-pCPT-cGMP (lanes 2 and 3) for 30 min. VASP phosphorylation on Ser was assessed using a that fluid shear stress induces fos phosphorylation site-specific antibody; a duplicate Western blot was probed with an anti--tubulin anti- body. D, MC3T3 cells were treated for 1 h with 10 M sodium nitroprusside, and c-fos, fra-1, and fra-2 mRNA family gene expression in osteo- levels were quantified by real time RT-PCR. E, MC3T3 cells were treated for 1 h with either cGMP (100 M blasts via NO/cGMP/PKG signal- 8-pCPT-cGMP), calcium ionophore (0.3 M A23187), or both agents, and fos family gene expression was measured as in B. ing, we examined whether gene induction occurred via PKG I, PKG Fluid shear stress rapidly increases the intracellular calcium II, or both. Because there are no specific inhibitors for PKG I concentration in MC3T3 cells and primary osteoblasts/osteo- versus II, we used an siRNA approach. In MC3T3 cells trans- cytes through calcium release from intracellular stores and cal- fected with an siRNA targeting a C-terminal PKG I sequence MAY 29, 2009• VOLUME 284 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 14801 PKG and Shear Stress-induced Gene Expression (siRNA PKG-1, targeting both PKG I and PKG I splice variants), PKG I mRNA levels were reduced by 68%, compared with the levels found in control siRNA-transfected cells, whereas PKG II mRNA levels were unaffected (Fig. 5A; PKG I and II mRNA levels are shown in black and gray bars, respectively). Using an antibody specific for the common C terminus of PKG I and I,we found that PKG I protein in cytoso- lic extracts was reduced in propor- tion to the mRNA knockdown (Fig. 5B, top panel; the 2nd panel shows a duplicate Western blot probed with a tubulin antibody to demonstrate equal loading). In MC3T3 cells transfected with siRNA sequences targeting PKG II (siRNAs PKG-2a and -2b), PKG II mRNA levels were reduced by 60 and 68%, respectively, compared with control siRNA- transfected cells, but PKG I mRNA levels were not altered (Fig. 5A). PKG II protein levels in the mem- branes of cells transfected with con- trol or PKG I-specific siRNAs were similar, but levels were below detec- tion in cells transfected with PKG II-specific siRNAs (Fig. 5B, 3rd panel; loading was shown by re-probing the blot with an antibody specific for 3 integrin, as shown in the bottom panel). We measured similar PKG activities in membrane preparations of control siRNA- and PKG I siRNA-transfected MC3T3 cells and significantly reduced activ- ity in PKG II siRNA-transfected cells (Fig. 5C). The residual mem- brane-bound PKG activity in PKG II siRNA-transfected cells is likely FIGURE 5.EffectofsiRNA-mediatedPKGIorPKGIIknockdownonshear-andcGMP-inducedc-fos,fra-1,fra-2, explained by the higher sensitivity andfosB/fosBmRNAexpression. MC3T3 cells were transfected with siRNAs specific for GFP (control), or received siRNAs targeting sequences in PKG I (siRNA PKG-1) or PKG II (siRNAs PKG-2a and -2b) as described under “Experi- of the activity assay compared with mental Procedures.” A, at 48 h after transfection, mRNA levels of PKG I (black bars) and PKG II (gray bars) were Western blotting. Because siRNA quantified by real time PCR; levels were normalized to gapd mRNA, which was not affected by any of the siRNAs. knockdown of PKG I did not affect Relative PKG mRNA levels measured in GFP (control) siRNA-transfected cells were assigned a value of 100%. *, p 0.05 for the comparison between control siRNA and PKG siRNA-treated cells. B, in parallel experiments, cells were membrane-associated PKG activity, extracted by Dounce homogenization and fractionated by differential centrifugation; cytosolic fractions (upper two and Western blots failed to detect panels) and membrane fractions (lower two panels) were analyzed by SDS-PAGE/Western blotting using an antibody specific for the C terminus of PKG I common to PKG I and I(upper panel), an anti--tubulin antibody (2nd panel), an PKG I in membrane preparations, antibody specific for PKG II (3rd panel), and an anti-3 integrin antibody (lowest panel). C, siRNA-transfected cells we conclude that membrane-asso- were fractionated as described in B, and PKG activity was determined in the membrane fractions as described under ciated PKG activity in MC3T3 cells “Experimental Procedures.” *, p 0.05 for the comparison between control siRNA and PKG siRNA-treated cells. D, cells transfected with the siRNAs targeting GFP, PKG I, or PKG II (PKG-2a siRNA) were kept either under static represents predominantly PKG II, conditions or were subjected to fluid shear stress for 20 min. Cells were harvested 60 min after the onset of flow, and which is consistent with results in c-fos, fra-1, and fra-2 mRNA levels were determined by quantitative RT-PCR; relative mRNA levels (normalized to other cell types (40, 53, 66, 67). gapd) measured in GFP siRNA-transfected static control cells were assigned a value of 1. E, cells transfected with the indicated siRNAs were either mock-treated or were treated with 50M 8-pCPT-cGMP for 1 h, and c-fos, fra-1, or fra-2 When we examined the effects of mRNA levels were determined by quantitative RT-PCR. Relative mRNA levels (normalized to gapd) measured in GFP siRNA-mediated down-regulation siRNA-transfected mock-treated cells were assigned a value of 1. *, p 0.05 for the comparison between GFP and PKG II siRNA-treated cells (D and E). of PKG I and PKG II on shear-in- 14802 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009 PKG and Shear Stress-induced Gene Expression duced fos family gene expression in MC3T3 cells, we were sur- prised to find that knockdown of PKG I had no significant effect on gene expression, whereas knockdown of PKG II significantly diminished fluid shear stress-induced c-fos, fra-1, and fra-2 mRNA expression (Fig. 5D; results for PKG-2a siRNA are shown, but similar results were obtained with PKG-2b siRNA). We also examined 8-pCPT-cGMP-induced fos family gene expression in siRNA-transfected MC3T3 cells. Neither basal nor cGMP-induced c-fos, fra-1 or fra-2 mRNA levels were altered in PKG I siRNA-transfected cells, whereas cGMP-in- duced mRNA levels were severely reduced in cells transfected with the PKG II-specific siRNA (Fig. 5E). Similar results were obtained for fosB/fosB (supplemental Fig. 3). Thus, fluid shear stress- and cGMP-induced c-fos, fra-1, fra-2 and fosB/fosB mRNA expression in osteoblasts requires PKG II but not PKG I. To confirm these results, we used a second PKG II-specific siRNA (PKG-2b) and an adenoviral vector encoding rat PKG II, which is resistant to the effects of this siRNA. In MC3T3 cells transfected with the mouse PKG II-specific siRNA, infection with rat PKG II virus, but not with a control LacZ virus, restored cGMP-induced fos family gene expression to the levels found in control GFP siRNA-transfected cells (Fig. 6A). Viral transduc- tion of PKG II in control siRNA-transfected cells did not fur- ther enhance cGMP-induced c-fos or fra-2 mRNA expression, suggesting that endogenous PKG II levels, although low, were not rate-limiting. However, cGMP stimulation of fra-1 mRNA was enhanced in cells with elevated PKG II levels (Fig. 6A, p 0.05 for the comparison of GFP siRNA-treated cells infected FIGURE 6. Rescue of PKG II siRNA-transfected cells with a virus encoding with LacZ versus PKG II virus). Viral PKG II expression was siRNA-resistant PKG II. A, MC3T3 cells were transfected with either GFP siRNA or the mouse PKG II-specific siRNA (PKG-2b), and 24 h later, cells were demonstrated by Western blotting of membranes prepared infected with either control virus encoding -galactosidase (LacZ) or virus from PKG II virus-infected MC3T3 cells (Fig. 6B; again, endog- encoding siRNA-resistant rat PKG II as indicated. Forty eight hours later, cells enous PKG II levels in PKG II siRNA-transfected cells were were either mock-treated or were treated with 8-pCPT-cGMP for 1 h, and fos family gene expression was determined as described in Fig. 5E.*, p 0.05 for below detection). the comparison between GFP versus PKG II siRNA-treated cells infected with Induction of c-fos, fra-1, fra-2, and fosB/fosB mRNA Expres- LacZ virus and the comparison between PKG II siRNA-treated cells infected with LacZ versus PKG II virus. B, cells were transfected with GFP siRNA (lanes 1, sion by Fluid Shear Stress Is MEK/Erk-dependent—Mechanical 2, 5, and 6) or PKG-2b siRNA (lanes 3, 4, 7, and 8) and were infected with LacZ stimulation enhances osteoblast proliferation via activation of virus (odd lanes) or PKG II virus (even lanes) as described in A. Levels of endog- the MEK/Erk pathway (20, 54, 62, 68). Whether MEK inhibition enous murine PKG II and virally expressed rat PKG II were examined by West- ern blotting of membrane and cytosolic proteins. prevents c-fos mRNA expression appears to depend on the type of mechanical stimulus and/or the source of osteoblasts, with variable results also reported for the effect of p38 MAPK inhib- fluid shear stress-induced fosB mRNA expression was reported itors on mechanically induced c-fos expression (20–22, 24, 54). in primary rat osteoblasts (54). We therefore investigated the effects of MEK and p38 inhibi- Fluid Shear Stress-induced Erk Activation Requires NO/ tors in fluid shear-stressed osteoblasts. cGMP/PKG II Signaling—Because fluid shear stress induction We found that fluid shear stress rapidly increased Erk1/2 of the four fos family genes required signaling through phosphorylation on a site that correlates with Erk1/2 activation NO/cGMP/PKG II and the MEK/Erk pathway, we asked (Fig. 7A shows results in MC3T3 cells, but similar results were whether Erk activation occurred through NO/cGMP/PKG II. obtained in hPOBs and UMR106 cells). Maximal induction of Pretreating osteoblasts with L-NAME, ODQ, or (R )-8-pCPT- Erk phosphorylation after 10 min of shear stress was 11.4-fold PET-cGMPS prevented shear-induced Erk1/2 phosphorylation compared with sham-treated cells. Pretreating cells with the in MC3T3 cells (Fig. 8A); similar results were obtained in MEK inhibitor U0126 blocked Erk phosphorylation and almost hPOBs (supplemental Fig. 4A; L-NAME not shown in hPOBs). completely prevented shear induction of c-fos, fra-1, fra-2, and Fluid shear stress-induced Erk phosphorylation was abolished fosB/fosB mRNAs (Fig. 7, B and C; U0126 inhibition of fosB/ in MC3T3 cells transfected with a PKG II-specific siRNA, but fosB mRNA induction was measured by semi-quantitative transfection of a PKG I-specific siRNA was without effect (Fig. RT-PCR; data not shown). The p38 inhibitor SB203580, how- 8B; results similar to those shown for PKG-2a siRNA were ever, did not affect Erk or fos family genes (Fig. 7, B and C; obtained with PKG-2b siRNA). Thus, Erk activation by shear SB203580 inhibited UV stress-induced p38 activation in con- stress, like shear-induced fos family gene expression, required trol experiments; data not shown). A similar effect of U0126 on NO/cGMP activation of PKG II. MAY 29, 2009• VOLUME 284 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 14803 PKG and Shear Stress-induced Gene Expression FIGURE 7. Induction of c-fos, fra-1, and fra-2 mRNA expression by fluid shear stress is MEK/Erk-dependent. A, serum-deprived MC3T3 cells were kept under static conditions for 30 min (sham-treated, lane 1) or were subjected to laminar flow for 5 min (lane 2), 10 min (lane 3), or 20 min (lanes 4 and 5); cells in lane 5 were kept in the flow chamber for an additional 10 min after the 20- min exposure to flow. Cell lysates were analyzed by Western blotting using a phospho-Erk1/2-specific antibody (clone E-4, upper panel); duplicate blots were probed with an antibody recognizing Erk1/2 irrespective of their phosphoryl- ation state (lower panel). The bar graph below summarizes results of three independent experiments; phospho-Erk1 bands were scanned, and the intensity of the band found in sham-treated cells was assigned a value of 1. B, serum-deprived MC3T3 cells were preincubated for1hinthe flow chamber under static conditions with either culture medium alone or with medium containing 10 M U0126 or 10 M SB203580, and cells were exposed to laminar flow for 10 min; Erk phosphorylation was assessed as described in A, with phospho-Erk levels in shear-stressed cells incubated with media alone assigned a value of 100%. C, cells were treated as described in B; they were exposed to laminar flow for 20 min and were harvested 10 min later for determination of c-fos, fra-1, fra-2, and gapd mRNA levels by quantitative RT-PCR, as described in Fig. 1B.*, p 0.05 for the comparison between cells treated with U0126 (U) versus control cells receiving no drug (in B and C). S, SB203580. NO/cGMP Activation of PKG II Is Sufficient to Activate Erk1/2—We next examined whether direct PKG activation by 8-pCPT-cGMP affected Erk phosphorylation in osteoblasts. In serum-starved MC3T3 cells, 50 M 8-pCPT-cGMP increased Erk1/2 phosphorylation 7.6-fold at 10 min, with phospho-Erk levels returning to base line at 30 min (Fig. 9A; compara- ble results were obtained in hPOBs, shown in supplemental Fig. 4B). In cells treated with A23187 to increase intracellular calcium, Erk phospho- rylation increased to a similar extent as observed with 8-pCPT-cGMP, and the effect of both agents appeared additive (supplemental Fig. 2C). FIGURE 8. Fluid shear stress-induced Erk activation requires NO/cGMP/PKG II signaling. MC3T3 cells were These results are consistent with the serum-deprived for 24 h prior to the indicated treatments, and Erk phosphorylation was assessed as described in Fig. 7. A, cells were treated for1hwith4mML-NAME, 10 M ODQ, or 100 M (R )-8-pCPT-PET-cGMPS combined effects of calcium and (Rp-cGMPS) as indicated, prior to a 10-min exposure to laminar flow (lanes 2–5); cells in lane 1 were kept under cGMP on c-fos, fra-1, fra-2, and fosB/ static conditions (sham-treated). In the bar graph below, three independent experiments are summarized. p fosB mRNA expression (Fig. 4E and 0.05 for the comparison between control cells receiving no drug and cells treated with L-NAME, ODQ, or (R )-cGMPS. B, cells were transfected with control siRNAs specific for GFP or received siRNAs targeting PKG I p supplemental Fig. 2, A and B). (siRNA PKG-1) or PKG II (siRNA PKG-2a) as described in Fig. 5. Cells were either kept under static conditions () Inhibition of MEK by U0126 or or were exposed to laminar flow ( ) for 10 min. The bar graph summarizes two independent experiments. p 0.05 for the comparison between cells transfected with PKG-2a siRNA versus GFP siRNA. inhibition of PKG by (R )-8- 14804 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009 PKG and Shear Stress-induced Gene Expression FIGURE 9. NO/cGMP activation of PKG II is sufficient to activate Erk1/2. Erk phosphorylation was assessed in serum-deprived MC3T3 cells as described in Fig. 7A. A, cells were treated with 50 M 8-pCPT-cGMP for the indicated times. The bar graph summarizes three independent experiments. B, cells were preincu- bated for1hin medium alone or in medium containing 10 M U0126 or 10 M SB203580 as indicated, prior to receiving 50 M 8-pCPT-cGMP (cGMP) for 10 min; phospho-Erk levels in cells treated with cGMP alone were assigned a value of 100%. C, cells were transfected with a control siRNA specific for GFP (lanes 1– 4), or an siRNA targeting PKG I (siRNA PKG-1, lanes 5– 8), or PKG II (siRNA PKG-2a, lanes 9 –12) as described in Fig. 5 and were treated with 50 M 8-pCPT-cGMP for the indicated times. The bar graph on the right summarizes phospho-Erk1 levels measured in cells treated with cGMP for 5 min; p 0.05 was used for the comparison between cells transfected with PKG-2a siRNA versus GFP siRNA. D, cells were transfected with either GFP siRNA (lanes 1– 4) or the mouse PKG II-specific siRNA PKG-2b (lanes 5–10); 8 h later, cells were infected with control virus (LacZ, lanes 1, 2, 5, and 6), virus encoding siRNA-resistant rat PKG II (lanes 3, 4, 7, and 8), or virus encoding PKG I (lanes 9 and 10). Forty eight hours later, cells were treated with 8-pCPT-cGMP for 10 min. The bar graph on the right summarizes cGMP-induced phospho-Erk1 levels; *, p 0.05 for the comparison between LacZ and PKG II virus-infected cells transfected with PKG-2a siRNA. pCPT-PET-cGMPS blocked cGMP-induced Erk phospho- with a LacZ- or PKG I-expressing adenovirus was without rylation in MC3T3 cells (Fig. 9B). ODQ inhibited Erk phos- effect (viral expression of PKG I was confirmed by Western phorylation induced by the NO donor PAPA-NONOate, and blotting and activity assay; data not shown). We conclude that (R )-8-pCPT-PET-cGMPS inhibited cGMP-induced Erk cGMP activation of PKG II is necessary and sufficient to medi- phosphorylation in hPOBs (supplemental Fig. 4B). ate Erk activation in osteoblasts and that PKG I cannot replace Consistent with PKG II mediating cGMP effects on fos family PKG II in this function. gene expression (Figs. 5 and 6), siRNA-mediated suppression of PKG II prevented cGMP-induced Erk activation, whereas sup- DISCUSSION pression of PKG I was without effect (Fig. 9C). To confirm the We found that fluid shear stress induced a rapid and sus- role of PKG II in regulating Erk1/2, we transfected cells with the tained increase in c-fos, fra-1, fra-2, and fosB/fosB mRNA in mouse-specific siRNA PKG-2b and restored PKG II expression human primary osteoblasts, MC3T3, and UMR106 cells. These by infecting cells with adenovirus encoding rat PKG II. Viral fos family genes encode members of the AP-1 transcription fac- expression of PKG II restored cGMP-induced Erk phosphoryl- tor family that regulate osteoblast proliferation and differenti- ation in PKG-2b siRNA-transfected cells (Fig. 9D; viral expres- sion of rat PKG II is shown in Fig. 6B). In contrast, infecting cells ation (6, 8, 10, 11). We determined that mechanical stimulation MAY 29, 2009• VOLUME 284 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 14805 PKG and Shear Stress-induced Gene Expression ronal cells, PKG II, NO synthase, and sGC localize to special- ized synaptic membrane domains that contain glutamate receptors (66, 73). A similar co-localization of eNOS, sGC, and PKG II may occur in osteoblast membranes, near mechanore- ceptors that transduce biophysical stimuli into biochemical sig- nals. Possible mechanoreceptors include mechanosensitive calcium channels, G-proteins activated by membrane deforma- tion, and integrin adhesions and cytoskeletal structures (3, 5, 74). We previously showed in osteoblasts that PKG II phospho- rylates and inactivates glycogen synthase kinase-3, leading to increased DNA binding activity of CAAT enhancer-binding protein (C/EBP-); the latter cooperates with CREB in medi- ating cGMP regulation of c-fos (40, 75). In contrast, cGMP stimulation of interleukin-6 transcription in osteoblasts FIGURE 10. NO/cGMP and calcium signaling in osteoblast mechanotrans- requires PKG I (41). Thus, PKG I and II have different roles in duction. Osteoblast exposure to fluid shear stress leads to a rapid increase in osteoblasts, consistent with our finding that PKG II, but not intracellular calcium through activation of mechanosensitive and voltage- PKG I, mediates mechanotransduction. gated calcium channels (MSCC and VGCC, respectively), and possibly through calcium release from intracellular stores (17, 54, 62, 63, 69). An initial burst of Mice with a targeted deletion of PKG II and rats with a spon- NO synthesis may require calcium activation of NO synthase (NOS), but sus- taneous deletion in the PKG II gene develop dwarfism because tained NO synthesis is calcium-independent and may involve activation of NO synthase by phosphorylation (57, 59). NO activates sGC and the resulting of impaired chondroblast differentiation (38, 76). In chondro- cGMP activates membrane-bound PKG II, which phosphorylates substrates blasts, PKG II regulates the switch from proliferation to hyper- leading to activation of the MEK/Erk pathway. The MEK/Erk pathway trophic differentiation by inhibiting the transcription factor increases transcription of c-fos, fra-1, fra-2, and fosB/fosB through the follow- ing: (i) RSK-mediated phosphorylation and activation of the CREB; (ii) direct Sox9 and by phosphorylating and inactivating glycogen syn- phosphorylation of Elk that forms a complex with and activates serum-re- thase kinase-3; the latter suppresses -catenin degradation, sponse factor (SRF); and (iii) recruitment of AP-1 (Fos/Jun) complexes to the promoters (79). Calcium activates the MEK/Erk pathway through Ras/Raf acti- leading to increased differentiation (39, 77). Our results suggest vation of MEK, and can directly activate CREB through calmodulin-dependent that defective PKG II regulation of c-fos, fra-1, fra-2, and fosB/ protein kinase (CamK) (89). Inhibitors used in this study are indicated in gray. fosB in osteoblasts and/or chondroblasts may contribute to BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid. the developmental bone defects observed in PKG II-deficient of all four fos family genes was mediated by the NO/cGMP/PKG rodents. No obvious skeletal abnormalities have been observed II signaling pathway and required PKG II-dependent Erk acti- in PKG I-deficient mice, but their life span is too short to deter- vation (Fig. 10). mine whether PKG I contributes to post-natal skeletal homeo- One of the earliest effects of fluid shear stress on osteoblasts stasis (78). is an increase in the intracellular calcium concentration. In We found that fluid shear stress induction of c-fos, fra-1, both MC3T3 cells and human osteoblasts, the increase is fra-2, and fosB/fosB mRNAs required signaling through the detectable within seconds of flow onset, and is sustained for at MEK/Erk but not the p38 MAPK pathway. These findings are least several minutes (17, 63, 69). Increased intracellular cal- consistent with previous work showing that the MEK inhibitor cium leads to activation of constitutively expressed NO syn- U0126 largely prevents c-fos induction in MC3T3 cells exposed thases, and increased NO production in osteoblasts occurs to gravitational force and fosB/fosB induction in mechanically within 5 min of flow onset (55, 56, 70) (Fig. 2A). However, only stimulated primary rat osteoblasts, whereas the p38 inhibitor the initial burst of NO synthesis is calcium-dependent, whereas SB203580 has no effect (20, 24, 54). The MEK/Erk signal trans- the later sustained phase of NO production is calcium-indepen- duction pathway regulates transcription of all four fos family dent (57). Our results suggest that the sustained stimulation of genes by targeting common cis-acting promoter elements, i.e. NO synthase activity may occur through eNOS phosphoryla- the serum-response element, cAMP-response element, and tion by multiple kinases, as observed in fluid shear-stressed phorbol ester-response element(s) (AP-1-binding sites) (Fig. endothelial cells (59, 71). 10) (79). Erk phosphorylates the ternary complex factor Elk-1, We found that fluid shear stress stimulates osteoblasts to causing it to associate with and activate serum-response factor produce sufficient NO to increase the intracellular cGMP con- at the serum-response element, and Erk activation recruits centration and activate PKG, based on increased VASP phos- c-Jun/Fra-2-containing AP-1 complexes to the fra-1 and fra-2 phorylation on Ser , a site targeted by both PKG I and II. promoters (80–85). The Erk-activated kinases of the RSK fam- Using an siRNA approach, we showed that PKG II, but not PKG ily phosphorylate and activate CREB, which plays a major role I, is required for shear-induced c-fos, fra-1, fra-2 and fosB/fosB in fluid shear stress-induced transcription of fosB/fosB (54, 84, mRNA expression. This was surprising, because osteoblasts 86). Erk regulation of c-fos, fra-1, fra-2, and fosB/fosB expres- contain 5-fold more cytosolic PKG activity (PKG I) than sion may explain why Erk is essential for osteoblast growth and membrane-bound PKG activity (PKG II), and PKG I and II differentiation (20, 68, 87). share many substrates in vitro (72). However, PKG II may be The mechanism of Erk1/2 activation in mechanically stimu- uniquely situated for activation by shear-induced NO. In neu- lated osteoblasts is only partly understood. In fluid shear- stressed MC3T3 cells, Erk activation is calcium-dependent and S. Zhuang and R. Pilz, unpublished results. inhibited by chelation of intra- or extracellular calcium; 14806 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009 PKG and Shear Stress-induced Gene Expression 5. Rubin, J., Rubin, C., and Jacobs, C. R. (2006) Gene (Amst.) 367, 1–16 whether it requires calcium entry through mechano-sensitive 6. Wagner, E. F., and Eferl, R. (2005) Immunol. Rev. 208, 126–140 cation channels and/or L-type voltage-gated calcium channels 7. Ruther, U., Garber, C., Komitowski, D., Muller, R., and Wagner, E. F. is unclear (Fig. 10) (54, 62, 63). We found that fluid shear stress- (1987) Nature 325, 412–416 induced Erk1/2 phosphorylation was blocked by pharmacolog- 8. Grigoriadis, A. E., Schellander, K., Wang, Z. Q., and Wagner, E. F. (1993) ical inhibition of either NO/cGMP synthesis or PKG activity, J. Cell Biol. 122, 685–701 and siRNA experiments demonstrated that both shear- and 9. Jochum, W., David, J. P., Elliott, C., Wutz, A., Plenk, H., Jr., Matsuo, K., and Wagner, E. F. (2000) Nat. Med. 6, 980–984 cGMP-induced Erk activation was mediated by PKG II. Possi- 10. Sabatakos, G., Sims, N. A., Chen, J., Aoki, K., Kelz, M. B., Amling, M., ble targets of PKG II leading to Erk activation include MEK, Bouali, Y., Mukhopadhyay, K., Ford, K., Nestler, E. J., and Baron, R. (2000) focal adhesion kinase, and Src family kinases. Consistent with Nat. Med. 6, 985–990 our results, Kapur et al. (88) found that L-NAME blocked fluid 11. Eferl, R., Hoebertz, A., Schilling, A. F., Rath, M., Karreth, F., Kenner, L., shear stress-induced Erk phosphorylation in hPOBs, leading to Amling, M., and Wagner, E. F. (2004) EMBO J. 23, 2789–2799 decreased shear-induced DNA synthesis, but signaling down- 12. Kenner, L., Hoebertz, A., Beil, T., Keon, N., Karreth, F., Eferl, R., Scheuch, H., Szremska, A., Amling, M., Schorpp-Kistner, M., Angel, P., and Wag- stream of NO was not examined. 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Chem. 276, 7048–7054 14808 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 22 •MAY 29, 2009
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Published: May 1, 2009