Anti-inflammatory effect of MAPK phosphatase-1 local gene transfer in inflammatory bone loss

Abstract

Alveolar bone loss associated with periodontal diseases is the result of osteoclastogenesis induced by bacterial pathogens. The mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is a critical negative regulator of immune response as a key phosphatase capable of dephosphorylating activated MAPKs. In this study, rat macrophages transduced with recombinant adenovirus (Ad.)MKP-1 specifically dephosphorylated activated MAPKs induced by lipopolysaccharide (LPS) compared with control cells. Bone marrow macrophages from MKP-1 knockout (KO) mice exhibited higher interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and select chemokine compared with wild-type (WT) mice when stimulated by LPS. In addition, bone marrow cultures from MKP-1 KO mice exhibited significantly more osteoclastogenesis induced by LPS than when compared with WT mice. Importantly, MKP-1 gene transfer in bone marrow cells of MKP-1 KO mice significantly decreased IL-6, IL-10, TNF-α and chemokine levels, and formed fewer osteoclasts induced by LPS than compared with control group of cells. Furthermore, MKP-1 gene transfer in an experimental periodontal disease model attenuated bone resorption induced by LPS. Histological analysis confirmed that periodontal tissues transduced with Ad. MKP-1 exhibited less infiltrated inflammatory cells, less osteoclasts and less IL-6 than compared with rats of control groups. These studies indicate that MKP-1 is a key therapeutic target to control of inflammation-induced bone loss.

Figure 1

MKP-1 gene transfer specifically dephosphorylated MAPKs in rat macrophages after LPS stimulation. Rat macrophages (NR8383 cells) were transduced with Ad.MKP-1 (moi 300), or Ad.LacZ (moi 300), or treated with equal volume of HEPES buffered saline for 48 hours. Cells were either unstimulated or stimulated with 1μg/ml of LPS (from A. actinomycetemcomitans) for various time periods (10, 30, 60, 120, 180, and 300 minutes). The MKP-1, p-JNK (p54, p46), p-p38 MAPK, p-ERK (p44, p42), p-38 MAPK, and p-NF-κB (p65, p105) protein expression were examined by western blot assays. The p-38 MAPK served as a loading control. (a) MKP-1 protein expression in rat macrophages. (b) Phosphorylated MAPKs protein expression in rat macrophages. (c) Phosphorylated NF-κB p65 and p105 protein expression in rat macrophages. The data is a representative from three separate experiments.

Figure 2

MKP-1 gene transfer corrected the defective immune responses seen in macrophages from MKP-1 KO mice after LPS stimulation. Bone marrow macrophages from MKP-1 KO mice or WT mice were transduced with Ad.MKP-1 or control Ad.LacZ (moi=300) for 48 hours, cells were stimulated with 100ng/ml of LPS (from A. actinomycetemcomitans) for 24 hours. Cytokine expression in supernatant was evaluated by ELISA assay and was normalized by the protein concentration in cell lysates. The data represent the average of three separate experiments. (a) IL-6 expression in mouse bone marrow macrophages (n=4, **p<0.01, ***p<0.001). (b) TNF- α expression in mouse bone marrow macrophages (n=4, ***p<0.001). (c) IL-10 expression in mouse bone marrow macrophages (n=4, **p<0.01, ***p<0.001). (d) CXCL1 expression in mouse bone marrow macrophages (n=4, *p<0.05, ***p<0.001).

Figure 3

MKP-1 gene transfer inhibited osteoclasts formation in bone marrow (BM) cells from MKP-1 KO mice after LPS stimulation. BM cells were extracted from either eight-week-old MKP-1 KO mice or WT mice. Cells were transduced with Ad.MKP-1 or control Ad.LacZ (moi 25) for 48 hours and then incubated with MEM-alpha medium containing 10% FBS, 2 mM glutamine, 50ng/ml recombinant mouse M-CSF (R&D systems) with or without LPS (1μg/ml, from A. actinomycetemcomitans) for 8 days. Osteoclasts were stained by tartrate resistant acid phosphatase (TRAP) staining. (a) Multinucleated osteoclasts (arrows shown) in bone marrow cells from MKP-1 KO mice and WT mice. (b) Number of osteoclasts (more than three nuclei) per well (96-well plate) in bone marrow cells from MKP-1 KO mice and WT mice (n=4, **p<0.01, ***p<0.001).

Figure 4

LacZ gene expression in rat gingival tissues after Ad.LacZ injection. Ad.LacZ (1×10⁹ pfu, or 5×10⁸ pfu in 4 μl) was injected in the gingival tissues of eight-week-old of male Sprague-Dawley rats (21 rats/group). Gingival tissues were harvested at 3, 21, and 28 days after Ad.LacZ injection (7 rats/group). LacZ gene expression was evaluated by β-galactosidase staining in frozen tissue sections. The scale bars represent 200 μm. Pictures represent average LacZ gene expression in each group.

Figure 5

MKP-1 gene transfer alleviated bone resorption in rats after LPS challenge. Eight-week-old of male Sprague-Dawley rats (17 rats/group) were injected either Ad.MKP-1, or Ad. LacZ (1×10⁹ pfu in 4 μl), or HEPES buffered saline (4 μl). Forty-eight hours after the adenovirus injection, the rats were injected with 2μl of either 20 μg of LPS (from A. actinomycetemcomitans) or PBS three times a week for four weeks. (a) Representative microcomputed tomography images of rat maxillae from indicated treatment groups. (b) Volumic analysis of bone loss levels (n=7 for PBS groups, n=10 for LPS groups, *p<0.05).

Figure 6

MKP-1 gene transfer reduced inflammatory infiltration and osteoclastogenesis in rats after LPS challenge. Rat tissue sections (7 μm) were stained by hematoxylin & Eosin (H.E.). Histological appearance of lower magnification (40 x) and higher magnification (400 x) of periodontal tissues from rat maxillae injected with PBS or LPS. Arrows show multinucleated osteoclasts near the surface of alveolar bone. Scale bars represent 1500 μm for lower magnification (40 x), and 90 μm for higher magnification (400 x).

Figure 7

MKP-1 gene transfer attenuated osteoclastogenesis induced by LPS. Rat tissue sections (7 μm) were stained for tartate-resistant acid phosphatase (TRAP). (a) TRAP staining of representative tissue sections of rat maxillae from indicated treatment groups. TRAP-positive (TRAP⁺) osteoclasts were stained red. The scale bars represent 200 μm. (b) Number of TRAP⁺ cells in each tissue section. Horizontal bar indicates mean TRAP⁺ cell counts (n=7 for PBS groups, n=10 for LPS groups, *** p<0.001).

Figure 8

MKP-1 gene transfer decreased IL-6 expression in rat tissues after LPS challenge. Rat tissue sections (7 μm) were immunohistochemically stained for MKP-1 and IL-6. (a) MKP-1 and IL-6 expression of representative tissue sections of rat maxillae from indicated treatment groups. The scale bars represent 200 μm. (b) Histological scores of IL-6 expression in tissue sections. (n=7 for PBS groups, n=10 for LPS groups).