Elsevier

Matrix Biology

Volume 24, Issue 7, October 2005, Pages 478-488
Matrix Biology

MKK3/6—p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1β and TNF-α in immortalized periodontal ligament fibroblasts

https://doi.org/10.1016/j.matbio.2005.06.006Get rights and content

Abstract

Matrix metalloprotease-13 (MMP-13) or collagenase-3 is involved in a number of pathologic processes such as tumor metastasis and angiogenesis, osteoarthritis, rheumatoid arthritis and periodontal diseases. These conditions are associated with extensive degradation of both connective tissue and bone. This report examines gene regulation mechanisms and signal transduction pathways involved in Mmp-13 expression induced by proinflammatory cytokines in periodontal ligament (PDL) fibroblasts. Mmp-13 mRNA expression was increased 10.7 and 9.5 fold after stimulation with IL-1β (5 ng/mL) and TNF-α (10 ng/mL), respectively. However, inhibition of p38 MAPKinase with SB203580 resulted in significant (p < 0.001) induction (23.2 and 18.1 fold, respectively) of Mmp-13 mRNA as assessed by real time PCR. Negative regulation of IL-1β induced Mmp-13 expression was confirmed by inhibiting p38 MAPK gene expression with siRNA. Transient transfection of dominant negative forms of MKK3 and MKK6 also resulted in increased levels of Mmp-13 mRNA after IL-1β stimulation. Mmp-13 mRNA expression induced by TNF-α was decreased by JNK and ERK inhibition. Western blot and zymogram analysis indicated that Mmp-13 protein expression induced by the proinflammatory cytokines were also upregulated by inhibition of p38 MAPK. Reporter gene experiments using stable cell lines harboring 660-bp sequence of the murine Mmp-13 proximal promoter indicated that transcriptional mechanisms were at least partially involved in this negative regulation of Mmp-13 expression by p38 MAPK and upstream MKK3/6. These results suggest a negative transcriptional regulatory mechanism mediated by p38 MAPK and upstream MKK3/6 on Mmp-13 expression induced by proinflammatory cytokines in PDL fibroblasts.

Introduction

Degradation of extracellular matrix (ECM) components is one of the main processes in a number of physiological and pathological events. Matrix metalloproteases (MMPs) are a family of neutral proteases responsible for degrading components of the extracellular matrix. MMP-13 was originally identified in human breast carcinomas (Freije et al., 1994), and is currently related to various other pathological conditions and processes associated with extracellular matrix degradation and bone resorption, such as squamous cell carcinomas, osteoarthritis, rheumatoid arthritis, tumor metastasis into bone and destructive periodontal disease (Kiili et al., 2002). Another common characteristic in those conditions is the presence of an immune-inflammatory reaction with high levels of proinflammatory cytokines. These high levels of cytokines can induce MMP production and secretion by resident cells such as chondrocytes (Ahmed et al., 2003, Liacini et al., 2002), endothelial cells (Zaragoza et al., 2002), fibroblasts (Solis-Herruzo et al., 1999), osteoblasts (Varghese and Canalis, 2003, Uchida et al., 2000). In support of MMP-13 potential role in destructive periodontal disease, increased levels of MMP-13 have been detected in diseased human periodontal tissues (Kiili et al., 2002, Uitto et al., 1998, Tervahartiala et al., 2000, Ejeil et al., 2003). In vivo and in vitro data suggests that MMP-13 expression may be associated with alveolar bone resorption in humans (Ma et al., 2000) and mice (Kusano et al., 1998). In vitro, MMP-13 expression was induced in gingival fibroblasts by TGF-β (Leivonen et al., 2002, Ravanti et al., 1999). Epithelial cells from the gingival sulcus of periodontitis-affected patients also expressed MMP-13 (Tervahartiala et al., 2000).

The periodontal ligament (PDL) is a highly vascularized and cellularized connective tissue attaching the root of the teeth to the surrounding alveolar bone. The fibroblastic cells (PDL cells) in this tissue present an osteoblast-like phenotype, including alkaline phosphatase activity and expression of osteocalcin. However, in the presence inflammatory cytokines, such as IL-1β, the PDL cells undergo transient phenotypic changes, losing their osteoblast-like characteristics (Agarwal et al., 1998) and expressing genes that are associated with catabolic processes, including bone resorption, which is supported by studies on the mechanism of orthodontic movement of teeth (Domon et al., 1999, Kanzaki et al., 2002). Other in vivo studies (Takahashi et al., 2003) have shown that MMP-13 in periodontal ligament cells during experimental orthodontic movement was higher on the compression side, which is associated with bone resorption. A previous study (Chang et al., 2002) suggested that MMP-2 was the main type of MMP produced by human periodontal ligament fibroblasts stimulated with IL-1α, but recently MMP-13 expression was shown in the same cell type upon stimulation with IL-1α (Noguchi et al., 2005) and also with TNF-α (Nishikawa et al., 2002). However, even though regulation of MMP-13 expression has been studied in different cell types, no studies to date have addressed intracellular signaling and molecular regulation mechanisms on PDL fibroblasts. Literature reports suggests that MMP-13 gene regulation is very complex, involving multiple signaling pathways (Liacini et al., 2003, Lechuga et al., 2004) as well as both transcriptional (Varghese and Canalis, 2003, Mengshol et al., 2001, Shah et al., 2004) and post-transcriptional mechanisms (Rydziel et al., 2004). Furthermore, regulation of MMP-13 appears also to depend on the cell type (Uchida et al., 2000) and the nature of the extracellular stimulation (Yang et al., 2004). This investigation indicates that p38 MAPKinase negatively regulates IL-1β and TNF-α-induced Mmp-13 expression at the mRNA and protein levels in mouse periodontal ligament cells by transcriptional mechanisms.

Section snippets

Inhibition of p38 MAPK and upstream MKK3 and MKK6 increases Mmp-13 mRNA and protein expression induced by IL-1β and TNF-α

Preliminary experiments indicated both the dose of cytokines (IL-1β: 5 ng/mL; TNF-α: 10 ng/mL) and stimulation period (8 h) associated with the highest level of Mmp-13 mRNA expression (Fig. 1), which were used for additional experiments. The same period of stimulation was reported to give the greatest response in MMP-13 induction in human chondrosarcoma cell line (Mengshol et al., 2000) and in rat osteoblastic cells (Varghese and Canalis, 2003), whereas the peak of TNF-α-induced MMP-13 mRNA was

Discussion

Regulation of MMP-13 is very complex, varying with the extracellular stimuli and cell type. We used mouse periodontal ligament fibroblasts that were immortalized by virus transformation. Even though these cells were previously characterized and shown to express genes that are normally expressed by ‘wild type’ murine PDL cells(D'Errico et al., 1999), the effect of viral transformation on Mmp-13 gene expression and regulation is not known. On the other hand, the use of immortalized cells might

Cells and materials

Mouse periodontal ligament (PDL) fibroblasts immortalized with SV40 large T antigen were obtained from Dr. Martha Somerman (University of Washington, Seattle, WA). These cells were cultured in DMEM supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal bovine serum and maintained in a humidified atmosphere at 37 °C and 5% CO2. Mouse PDL cell used were previously characterized for expression of genes normally expressed by primary PDL cells, including bone

Acknowledgments

This work was supported by the following grants: CAPES 0192/01-3, NIDCR R03 DE14460-01A1, and DOD W81XWH-05-0075. The authors also would like to thank Dr. Xinsheng Zhu (SUNY at Buffalo) for his suggestions and help in the beginning of this study.

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