Mechanical Strain Induces Collagenase-3 (MMP-13) Expression in MC3T3-E1 Osteoblastic Cells

Abstract

Mechanical strain plays a crucial role in bone remodeling during growth and development and healing of bone besides systemic and local factors. One of the major factors involves in remodeling process is matrix metalloproteinases (MMPs) such as MMP-13 that has been shown to degrade the native interstitial collagens in several tissues. To study how mechanical strain affects extracellular matrix degradation by MMP-13, a biaxial strain was applied to MC3T3-E1 osteoblastic cells plated onto a collagen-coated flexible elastic membrane. The MMP-13 protein and mRNA expression were determined by Western blotting and reverse transcriptase-PCR, respectively. The zymographic activities of MMP-13 increased dramatically at 30 min, reached a peak by 2-fold at 1 h, and maintained up to 4 h. Moreover, the MMP-13 and c-fos mRNA expressed at 5 min, increased to 2.8-and 3-fold at 1 h, respectively, and gradually declined thereafter. Cycloheximide and actinomycin D did not inhibit the MMP-13 and c-fos mRNA expression, suggesting that such expression does not require de novo protein synthesis and not change their stabilities. To investigate which of the mitogen-activated protein kinase (MAPK) pathways involves in the expression of MMP-13, inhibitors such as PD98059, SB203580, and SP600125 were used. However, only PD98059 (an inhibitor of MEK1/2 activation) inhibited MMP-13 and c-fos gene expression; the result was further substantiated by transfecting with the dominant negative mutants of MEK1/2 (MEK K97R) and ERK2. Taken together, our results showed that mechanical strain induces the MMP-13 expression through MEK-ERK signaling pathway to regulate mechanical adaptation.Bone is constantly remodeled throughout life to meet the functional demands of its physiological and mechanical environment (1-4). Remodeling of bone is regulated by a wide variety of systemic and local factors such as 1␣,25-dihydeoxyvitamin D 3 , parathyroid hormone, calcitonin, sex hormones, prostaglandin, transforming growth factor-␤, bone morphogenetic protein, and insulin-like growth factors. It is also a well known fact during growth and in the adult skeleton, a moderate level of mechanical loading is considered essential for maintaining and adaptation to the physiological bone remodeling (4, 5). In particular, mechanical loading on osteoblasts in vitro has been demonstrated to increase prostaglandin release (6), stimulate cell division (7), alter collagen synthesis (8), and promote collagenase activity (9).The remodeling of the mineralized connective tissue involves coupling of the degradation of the extracellular matrix (ECM) 1 with the synthesis of new matrix components. Although osteoblasts have been implicated only in bone formation, in response to resorption stimulators, they may cease synthesis of collagen and start secreting neutral proteinases such as collagenase (10 -12). These collagenases may degrade the unmineralized osteoid layer covering bone surfaces, leading to the exposure of the mineralized matrix to osteoclasts. On the other hand, recent findi...

Molecular pathways mediating mechanical signaling in bone

“…It has long been known to occur in endothelial cells, where not only ERK1/2 kinase, but also p38, BMK-1 and JNK are activated (Yan et al, 1999). In bone cells many groups have shown activation of ERK1/2 in particular (You et al, 2001;Jessop et al, 2002;Weyts et al, 2002;Yang et al, 2004;Plotkin et al, 2005).…”

“…As a result of increased eNOS expression, nitric oxide (NO) synthesis is enhanced. ERK1/2 activation by mechanical stress has been linked to other specific genes such as strain induction of collagenase-3 (Yang et al, 2004), as well as to proliferation (Boutahar et al, 2004). Both fosB and its spliced variant, deltafosB, which has been shown to stimulate increases in bone density in vivo, are induced by mechanical loading in mouse hind limb bone and by fluid shear stress in mouse calvarial osteoblasts (Inoue et al, 2004).…”

“…extracellular signal-regulated kinase MAPK mitogen-activated protein kinase Osteopontin (You et al, 2001) (Toma et al, 1997;Ziambaras et al, 1998) Collagenase-3 (Yang et al, 2004) Gene. Author manuscript; available in PMC 2013 June 20.…”

Ubiquitin ligase Cbl‐b acts as a negative regulator in discoidin domain receptor 2 signaling via modulation of its stability

“…Our previous molecular evidences indicated that DDR2 signaling can directly up-regulate the transcription of MMP-13 [21], a protein that is abundantly expressed by MC3T3-E1 cells [23]. Thus, MMP-13 was chosen as a molecular marker to evaluate the consequence of Cbl-b knockdown on DDR2 downstream events.…”

Multiphase and individual cell-based models of tumour growth

“…Moreover, it was found that many tumours are characterised by the formation of fibrotic tissue (Brewster et al 1990, Johnson 2001, Pujuguet et al 1996 or by degradation of ECM due to excessive production of MMP-13 (Yang 2004). Johnson (2001) argued that the formation of fibrosis can be due to several probably concurring reasons: increased de novo synthesis of ECM proteins, decreased activity of its degrading enzyme (MMP), and upregulation of the tissuespecific inhibitors of metalloproteinases (TIMP), that is, the molecules that make matrix degrading enzymes (MDEs) ineffective.…”