Loading-related regulation of transcription factor EGR2/Krox-20 in bone cells is ERK1/2 protein-mediated and prostaglandin, Wnt signaling pathway-, and insulin-like growth factor-I axis-dependent

Abstract

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression. In UMR106 cells, inhibition of β1/β3 integrin function had no effect on strain-related Egr2 expression, but it was inhibited by a COX2-selective antagonist and imitated by exogenous prostaglandin E2 (PGE2). This response to PGE(2) was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signaling, or LiCl had any effect on Egr2 mRNA expression, but it was increased by both insulin-like growth factor-1 and high, but not low, dose parathyroid hormone and exogenous Wnt-3a. The increases by strain, PGE2, Wnt-3a, and phorbol 12-myristate 13-acetate were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signaling pathways that constitute early responses of bone cells to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent "instructions" for adaptive (re)modeling is likely to depend upon their contextual activation, suppression, and interaction probably on more than one occasion.

FIGURE 1.

EGR2 is predicted to regulate loading-induced signaling networks involving ERK signaling. A, PASTAA analysis was used to identify the time points following loading in which EGR2 is most closely associated with the observed transcriptional changes. A significant EGR2 positional matrix signature was observed across the conserved regions within the −10-kb region of all the altered genes 3 and 12 h following loading (*, p < 0.001). The affinity scores for all the genes available at each time point are shown, indicating the greatest affinity scores at 8 h. For further analysis, an arbitrary threshold (horizontal line) was set at an affinity score of 0.1 to obtain a set of genes enriched for putative EGR2 target genes. This subset of 149 genes was used for subsequent functional analysis. Of these genes, 36% were up-regulated and 64% were down-regulated. B, cellular processes identified to be significantly enriched by Ingenuity pathway analysis software within this gene cluster are shown, suggesting potential functions for EGR2-regulated genes following loading. This was complemented by analysis of functional processes significantly enriched in this subset of genes using DAVID (david.abcc.ncifcrf.gov) revealing various gene ontology clusters with significantly altered components relating to the following: C, cytoskeleton and cell proliferation (12 genes differentially regulated in the cluster, represented by the horizontal axis); D, cell migration (5 genes); E, apoptosis (9 genes), and F, motility (5 genes). The number of genes pertaining to each GO term within each cluster is shown by the number of green boxes adjacent to the term, and black boxes represent genes within the cluster not yet associated with the GO term. Pathway analysis was also performed using Ingenuity Pathway Analysis. Given the small number of genes available, only the two most significantly altered pathways were studied. These pathways relate to cell death/endocrine system development (G) and cell movement/free radical scavenging (H).. Both pathways relate to ERK signaling. Genes in red are up-regulated, and those in green were down-regulated.

FIGURE 2.

Effect of loading on normalized Egr2 mRNA expression. A, total RNA was extracted at 3, 8, 12, and 24 h after a single period of dynamic axial loading of the mouse tibia in vivo and Egr2 mRNA expression levels measured by RT-qPCR. Normalized Egr2 mRNA expression levels are shown as fold difference compared with the nonloaded controls. B, primary osteoblast-like cells derived from C57BL/6 mouse long bones were seeded onto plastic strips and subjected to strain (3400 microstrain, 1 Hz, 600 cycles). Total RNA was extracted at 1 and 3 h after strain, and Egr2 mRNA expression levels were measured by RT-qPCR. Results are shown as mean ± S.E. **, p < 0.01 versus static at 1 h. C, UMR106 cells were seeded onto plastic strips and subjected to strain. Total RNA was extracted 1 h after strain, and Egr2 mRNA expression levels were measured by RT-qPCR. Results are shown as mean ± S.E. (B and C). ***, p < 0.001.

FIGURE 3.

Effect of strain on EGR2 protein localization and expression. A, Western blot for EGR2 from whole cell lysates was prepared from static and strained UMR106 cells at 0.25, 0.5, 1, 2, and 4 h after treatment. β-Actin was used as a control for equal protein loading. B, scanning densitometry was performed on Western blots from three independent experiments. Values shown are mean ± S.E. *, p < 0.05 versus its corresponding time point static control, paired one-tailed t test. C, UMR106 cells were seeded onto plastic strips, subjected to strain, and fixed 1 h later. The subcellular localization of EGR2 (green staining) was determined by immunocytochemistry and confocal microscopy. Nuclear accumulation of EGR2 in strained cells is also visible (white arrowheads). Scale bar, 20 μm.

FIGURE 4.

Effect of disintegrin and echistatin on strain-induced Egr2 expression. A, UMR106 cells were seeded onto plastic strips and pretreated with 100 nm echistatin (disintegrin against β1 and β3 integrins) overnight and then subjected to a single period of strain. Total RNA was extracted 1 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as means ± S.E. ***, p < 0.001 versus static. B, to show that echistatin was biologically effective in blocking the downstream effects of integrin signaling, UMR106 cells were pretreated with and without 100 nm echistatin overnight, trypsinized, and then cultured on a mixed substrate of collagen and fibronectin for 30 min to activate β1 and β3 integrin function. Nonadherent cells (−) were collected in ice-cold PBS, centrifuged, and lysed in denaturing lysis buffer, whereas the cells attached to the collagen/fibronectin substrate (+) were briefly washed in ice-cold PBS and lysed in denaturing buffer. Western blot for phosphorylated focal adhesion kinase (pFAK) was prepared from whole cell lysates. β-Actin was used as a control for equal protein loading.

FIGURE 5.

Effect of COX2 inhibitor on strain and the effect of exogenous PGE2 on Egr2 mRNA expression. A, UMR106 cells were seeded onto plastic strips and subjected to strain in the presence or absence of 1 μm NS398 (selective COX2 inhibitor). Total RNA was extracted 1 h after strain and RT-qPCR used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. ***¹, p < 0.001 versus static; ***², p < 0.001 versus strain. B, UM106 cells were seeded in tissue culture coated dishes and treated with 5 μm PGE2. Total RNA was extracted 0, 10, 20, and 50 min and 2, 4, and 6 h after treatment, and RT-qPCR used to measure normalized Egr2 mRNA expression levels. C, UMR106 cells were seeded on 66-mm dishes and treated with 5 μm PGE2 for 1 h in the presence or absence of 1 μm cycloheximide (CHX). Total RNA was extracted 1 h after PGE2 treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. *, p < 0.05 versus vehicle. **, p < 0.01 versus vehicle. ***, p < 0.001 versus cyclohexamide.

FIGURE 6.

Effect of PGE2 receptor inhibitors on PGE2-induced Egr2 mRNA expression. A, total RNA was extracted from UMR106 cells, and RT-PCR was used to amplify EP1, EP2, EP3, and EP4 receptor transcripts. B, UMR106 cells were seeded on 66-mm dishes and treated with exogenous PGE2 (5 μm) in the presence or absence of 30 μm AH6809 (EP1 and EP2 receptor antagonist). Total RNA was extracted 1 h after strain, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. ***¹, p < 0.001 versus vehicle; ***², p < 0.001 versus PGE2. C, UMR106 cells were seeded on 66-mm dishes and treated with exogenous PGE2 in the presence or absence of 30 μm AH23848 (EP4 receptor antagonist). Total RNA was extracted 1 h after strain, and RT-qPCR used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. *, p < 0.05 versus PGE2, ***, p < 0.001 versus vehicle, **, p = 0.01 versus strain.

FIGURE 7.

Effect of PKC and PKA signaling pathways on PGE2-induced Egr2 mRNA expression. A, UMR106 cells were seeded on 66-mm dishes and treated with 500 nm PMA (PKC agonist). Total RNA was extracted 0, 10, 20, and 50 min and 2, 4, and 6 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. B, UMR106 cells were seeded on 66-mm plastic dishes and treated with dibutyryl cyclic AMP (db-cAMP) (stable analog of cAMP). Total RNA was extracted 1 h after treatment. and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. *, p < 0.05 versus vehicle. UMR106 cells were seeded onto plastic strips and treated with 5 μm PGE2 in the presence or absence of 1 μm H89 (selective PKA inhibitor). Total RNA was extracted 1 h after strain, and RT-qPCR used to measure normalized Egr2 (C) and Igf-1 mRNA (D) expression levels. Results are expressed as mean ± S.E. *, p < 0.05 versus PGE2; **, p < 0.01 versus vehicle; ***, p < 0.001 versus PGE2.

FIGURE 8.

Effect of nitric oxide signaling pathways on Egr2 mRNA expression. UMR cells were seeded onto plastic strips and treated with 100 μm SNAP2 (NO donor). Total RNA was extracted, and RT-qPCR used to measure normalized Egr2 (A) and heme oxygenase (Ho-1) (B) mRNA expression at 1 and 2 h after treatment, respectively. Results expressed as mean ± S.E. ***, p < 0.001.

FIGURE 9.

Effect of estrogen signaling pathways on Egr2 mRNA expression. A, UMR106 cells were seeded on 66-mm plastic dishes and treated with 10⁻⁸ m estrogen, 10⁻⁷ m ICI 182,780 (separately and together), and 5 μm PGE2 as well as PGE2 in the presence or absence of estrogen and ICI 182,780. Total RNA was extracted 1 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. ***, p < 0.001 versus vehicle. B, total RNA was extracted from vehicle and ICI 182,780-treated C57BL/6 mouse (subcutaneous, 4 mg/kg/day for 21 days) long bones, and RT-qPCR was used to measure Egr2 mRNA expression levels. Results are expressed as mean ± S.E.

FIGURE 10.

Effect of Wnt signaling pathways on Egr2 mRNA expression. A, UMR106 cells were seeded on 66-mm dishes and treated with Wnt-3a (100 ng/ml). Total RNA was extracted 1 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. ***, p < 0.001. B, LRP5^G171V mouse long bone-derived primary osteoblast-like cells were seeded onto plastic strips and subjected to strain. Total RNA was extracted 1, 3, and 6 h after strain, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. *, p < 0.05 versus static. C, UMR106 cells were seeded on 66-mm dishes and treated with LiCl (10 mm). Total RNA was extracted 1 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. D, UMR106 cells were transiently transfected with TopFlash plasmid containing six TCF/LEF-binding sites driving the expression of luciferase. A control plasmid containing Renilla. luciferase gene under the control of a cytomegalovirus (CMV) promoter was transfected at the same time to control the transfection efficiency. Cells were treated with 10 mm LiCl and harvested 24 h later. Firefly luciferase activity was determined and normalized to Renilla. Data are compiled from three experiments and shown as mean ± S.E. ***, p < 0.005 versus vehicle.

FIGURE 11.

Effect of PTH on Egr2 mRNA expression. A, UMR106 cells were seeded on 66-mm dishes and treated with PTH (10⁻⁸ and 10⁻¹⁰ m). Total RNA was extracted 1 h after treatment and RT-qPCR used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. *, p < 0.05. B, UM106 cells were seeded in tissue culture-coated dishes and treated with 10⁻⁸ m PTH. Total RNA was extracted 0, 10, 20, and 50 min and 2, 4, and 6 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. C, UMR106 cells were treated with 10⁻⁸ m PTH in the presence or absence of 1 μm H89. Total RNA was extracted 1 h after PTH treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. *, p < 0.05 versus vehicle; *¹, p < 0.05 versus PTH.

FIGURE 12.

Effect of IGF-1 on Egr2 mRNA expression. UMR106 cells were seeded on 66-mm dishes and treated with IGF-1 analog (des-(1–3)-IGF-1, 50 ng/ml). Total RNA was extracted at 0, 10, 20, and 50 min and 2, 4, and 6 h after treatment, and RT-qPCR used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E.

FIGURE 13.

Effect of PD98059 on strain-, PGE2-, IGF-1-, Wnt-3a-, and PMA-induced Egr2 mRNA expression. UMR106 cells were seeded on 66-mm dishes and treated with the following: strain (A); PGE2 (5 μm) (B); IGF-1 (50 ng/ml) (C); Wnt-3a (100 ng/ml) (D), and PMA (500 nm) (E) in the presence and absence of PD98059 (30 μm) pretreatment (30 min). Total RNA was extracted at 1 h after treatment, and RT-qPCR was used to measure normalized Egr2 mRNA expression levels. Results are expressed as mean ± S.E. **, p < 0.01; ***, p < 0.001, a = versus vehicle and b = versus treatment (strain, PGE2, IGF-1, Wnt-3a, and PMA).

FIGURE 14.

Effect of strain, PGE2, PMA, and IGF-1 on Sost mRNA expression. A, UMR106 cells were seeded onto plastic strips and subjected to strain. Total RNA was extracted at 15 h after strain, and normalized Sost mRNA expression levels were measured by RT-qPCR. Normalized Sost mRNA expression levels are shown compared with the static controls. Results are expressed as mean ± S.E. ***, p < 0.001. B, UMR106 cells were seeded on 66-mm plastic dishes and treated with PGE2 (5 μm), PMA (500 nm), and IGF-1 (50 ng/ml). Total RNA was extracted at 0, 10, 20, and 50 min and 2, 4, and 6 h after treatment, and RT-qPCR was used to measure normalized Sost mRNA expression levels. Results are expressed as mean ± S.E.

FIGURE 15.

Effect of EGR2 siRNA on PGE2-induced Egr2, Sost, and Oc mRNA expression. UMR106 cells were transfected with EGR2 siRNA (100 nm), and 24 h later were treated with PGE2 (5 μm). A, total RNA was extracted at 1 and 6 h after PGE2 treatment and analyzed for normalized Egr2 and Sost mRNA expression levels using RT-qPCR. Results are expressed as mean ± S.E. **, p < 0.001 versus vehicle. ***, p < 0.001 versus control siRNA and PGE2 treatment. B, total RNA was extracted at 4 h after PGE2 treatment and analyzed for normalized osteocalcin mRNA expression levels using RT-qPCR. Results are expressed as mean ± S.E. **, p < 0.001 versus vehicle. ***, p < 0.001 versus control siRNA and PGE2 treatment.