Role of glucocorticoid-induced leucine zipper (GILZ) in inflammatory bone loss

<div><p>TNF-α plays a key role in the development of rheumatoid arthritis (RA) and inflammatory bone loss. Unfortunately, treatment of RA with anti-inflammatory glucocorticoids (GCs) also causes bone loss resulting in osteoporosis. Our previous studies showed that overexpression of glucocorticoid-induced leucine zipper (GILZ), a mediator of GC’s anti-inflammatory effect, can enhance osteogenic differentiation <i>in vitro</i> and bone acquisition <i>in vivo</i>. To investigate whether GILZ could antagonize TNF-α-induced arthritic inflammation and protect bone in mice, we generated a TNF-α-GILZ double transgenic mouse line (TNF-GILZ Tg) by crossbreeding a TNF-α Tg mouse, which ubiquitously expresses human TNF-α, with a GILZ Tg mouse, which expresses mouse GILZ under the control of a 3.6kb rat type I collagen promoter fragment. Results showed that overexpression of GILZ in bone marrow mesenchymal stem/progenitor cells protected mice from TNF-α-induced inflammatory bone loss and improved bone integrity (TNF-GILZ double Tg vs. TNF-αTg, n = 12–15). However, mesenchymal cell lineage restricted GILZ expression had limited effects on TNF-α-induced arthritic inflammation as indicated by clinical scores and serum levels of inflammatory cytokines and chemokines.</p></div

Primer sequences.

<p>Primer sequences.</p

Glucocorticoid-Induced Leucine Zipper (GILZ) Antagonizes TNF-α Inhibition of Mesenchymal Stem Cell Osteogenic Differentiation

<div><p>Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine that inhibits osteoblast differentiation while stimulating osteoclast differentiation and bone resorption. TNF-α activates MAP kinase pathway leading to inhibition of osterix (Osx) expression. TNF-α also induces the expression of E3 ubiquitin ligase protein Smurf1 and Smurf2 and promotes degradation of Runx2, another key transcription factor regulating osteoblast differentiation and bone formation. We showed previously that overexpression of glucocorticoid (GC)-induced leucine zipper (GILZ) enhances osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We and others also showed that GILZ is a GC effecter and mediates GC anti-inflammatory activity. In this study, we asked the question whether GILZ retains its osteogenic activity while functioning as an anti-inflammatory mediator. To address this question, we infected mouse bone marrow MSCs with retroviruses expressing GILZ and induced them for osteogenic differentiation in the presence or absence of TNF-α. Our results show that overexpression of GILZ antagonized the inhibitory effects of TNF-α on MSC osteogenic differentiation and the mRNA and protein expression of Osx and Runx2, two pivotal osteogenic regulators. Further studies show that these antagonistic actions occur via mechanisms involving GILZ inhibition of TNF-α-induced ERK MAP kinase activation and protein degradation. These results suggest that GILZ may have therapeutic potential as a novel anti-inflammation therapy.</p> </div

Effect of GILZ on TNF-α-induced ERK phosphorylation and Smurf expression.

<p>(A, B) MSC-GFP and MSC-GILZ cells were serum starved for 48 hr and then treated with TNF-α (1 ng/ml) for 15 min before harvesting. Equal amounts of total cell lysates were separated on SDS PAGE gel, transferred onto membrane and analyzed for levels of phosphorylated ERK1/2 (pERK). Levels of pERK were quantified with NIH Image J software and presented as a ratio of pERK/total ERK in B. Value from untreated MSC-GFP is arbitrarily set as 1. Experiments were repeated three times with similar results. Levels of overexpressed GILZ are also shown. Error bars indicate SD. α-tubulin, instead of β-actin, was used as a loading control in these experiments because β-actin and ERK have similar molecular weights. (C–F) RNA and protein samples from MSC-GFP and MSC-GILZ cells used in the experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone-0031717-g002" target="_blank">figure 2</a> were analyzed by real-time qRT-PCR and Western blot to show the levels of Smurf1 and Smurf 2 mRNA (C, D) and protein (E). Quantitative results of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone-0031717-g003" target="_blank">Fig. 3E</a> are shown as bar graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone-0031717-g003" target="_blank">Fig. 3F and G</a>, respectively. Data were normalized to β-actin and expressed as fold changes relative to control cells cultured in GM. These experiments were repeated three times with similar results. Error bars indicate SD. a, <i>p</i><0.01 (n = 3).</p

Effect of GILZ on TNF-α regulation of Osx and Runx2 expression.

<p>Real-time qRT-PCR and Western blot analyses of mRNA and protein expression. MSC-GFP and MSC-GILZ cells were cultured in OS with or without 1 ng/ml TNF-α (OST) for 8 days. At the end of treatment, one set of cells was harvested for real-time qRT-PCR (A, B) and a second set for Western blot (C) analyses of Osx and Runx2. Equal loading of samples in each lane is shown by the levels of β-actin. These experiments were performed at least three times with similar results. Quantitative results for levels of Osx and Runx2 protein are presented as bar graphs (D, E). Value of intensity for MSC-GFP cells cultured in regular DMEM growth medium (GM) is arbitrarily set as 1. Error bars indicate S.D. a, <i>p</i><0.01 (n = 3).</p

Characterization of TNF-GILZ Tg mice.

<p>(<b>A</b>) FACS analysis showing percentages of human TNF-α-positive hematopoietic (CD11b+) and mesenchymal (CD11b-) lineage cell populations in bone marrow of the TNF- α and TNF-GILZ double Tg mice. Three to four mice were used in each group and the experiment was repeated twice with similar results. (<b>B</b>) ELISA assays showing serum levels of mouse TNF-α in TNF and TNF-GILZ Tg mice. Each data point represents one mouse. (<b>C</b>) Clinical arthritis scores of TNF and TNF-GILZ Tg mice. The results are expressed as means ± S.D. Unpaired t-tests were performed for comparison.</p

Bone phenotype of TNF-GILZ Tg mice.

<p>(<b>A</b>) DXA analysis of femoral samples showing bone mineral density (BMD) and content (BMC) of 6-month-old mice. Each data point represents one individual mouse. (<b>B</b>) uCT analysis showing bone integrity and architecture parameters of TNF and TNF-GILZ Tg mice. (<b>C</b>) Representative re-constructed 3D uCT images (femurs) of TNF and TNF-GILZ Tg mice. Samples of GILZ Tg littermates were used as a reference for GILZ anti-TNF-α actions. Results are expressed as means ± S.D. ANOVA with Bonferroni post hoc tests were performed for comparison.</p

Serum cytokine and chemokine levels in TNF and TNF-GILZ Tg mice.

<p>(<b>A</b>) ELISA assay showing serum levels of RANKL and OPG, and the ration of OPG/RANKL in TNF and TNF-GILZ Tg mice. Six to eight mice were used in each group. The experiment was repeated twice with similar results. (<b>B</b>) Multiplex immunoassay showing serum levels of different cytokines and chemokines in TNF and TNF-GILZ Tg mice. Each data point is from one individual mouse.</p

GILZ and IL-6 expression in synoviocytes and tibia and paw tissues.

<p>Real time qRT-PCR analysis showing levels of GILZ (<b>A</b>) and IL-6 mRNA (<b>B</b>) in fibroblast like synoviocytes (FLS) and tibia and paw tissues as indicated. Data is presented as fold change. Value from TNF mice is arbitrarily set as 1. Three to four mice were used in each group and the experiment was repeated twice with similar results.</p

H&E histology and histomorphormetry analysis of bone and joint.

<p>(<b>A</b>) Representative images of H&E stained femur of TNF and TNF-GILZ Tg mice (enlarged boxed areas are shown on right). (<b>B</b>) Representative IHC staining images of femurs. Boxed areas are enlarged (right) and arrows indicate osteocalcin-positive osteoblast cells. GP: growth plate; E: endocortical bone. (<b>C</b>) Bar graph showing quantified results of A. A Bioquant osteo image analysis system (Bioquant, Nashville, TN) was used to count the number of osteoblasts and measure the oasteoblast covered area. A defined region of interest was established ~0.5mm proximal to the distal growth plate and extended a further 0.5mm, all within the endocortical edges at 50x magnification. A total of 4 to 5 samples were analyzed for each group. (<b>D</b>) Representative histological images of paw-joint of TNF and TNF-GILZ Tg mice (20x). C: Cartilage damage; R: Bone resorption; I: inflammation; O: Osteophyte. Scale bar = 100μm.</p