E74-like factor 3 (ELF3) impacts on matrix metalloproteinase 13 (MMP13) transcriptional control in articular chondrocytes under proinflammatory stress.

Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position -78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1ß stimulation in chondrocytes, and the IL-1ß-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3(-/-) mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1ß-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.

GADD45beta enhances Col10a1 transcription via the MTK1/MKK3/6/p38 axis and activation of C/EBPbeta-TAD4 in terminally differentiating chondrocytes.

GADD45beta (growth arrest- and DNA damage-inducible) interacts with upstream regulators of the JNK and p38 stress response kinases. Previously, we reported that the hypertrophic zone of the Gadd45beta(-/-) mouse embryonic growth plate is compressed, and expression of type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13) genes is decreased. Herein, we report that GADD45beta enhances activity of the proximal Col10a1 promoter, which contains evolutionarily conserved AP-1, cAMP-response element, and C/EBP half-sites, in synergism with C/EBP family members, whereas the MMP13 promoter responds to GADD45beta together with AP-1, ATF, or C/EBP family members. C/EBPbeta expression also predominantly co-localizes with GADD45beta in the embryonic growth plate. Moreover, GADD45beta enhances C/EBPbeta activation via MTK1, MKK3, and MKK6, and dominant-negative p38alphaapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45beta and C/EBPbeta on the Col10a1 promoter. Importantly, GADD45beta knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPbeta binding to the Col10a1 promoter in vivo, indicating that GADD45beta influences the transactivation function of DNA-bound C/EBPbeta. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPbeta is the target of the GADD45beta-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45beta via the MTK1/MKK3/6/p38 axis to C/EBPbeta-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.

Inducible knockout of CHUK/IKKα in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis.

CHUK/IKKα contributes to collagenase-driven extracellular matrix remodeling and chondrocyte hypertrophic differentiation in vitro, in a kinase-independent manner. These processes contribute to osteoarthritis (OA), where chondrocytes experience a phenotypic shift towards hypertrophy concomitant with abnormal matrix remodeling. Here we investigated the contribution of IKKα to OA in vivo. To this end, we induced specific IKKα knockout in adult chondrocytes in AcanCreERT2/+; IKKαf/f mice treated with tamoxifen (cKO). Vehicle-treated littermates were used as wild type controls (WT). At 12 weeks of age, WT and cKO mice were subjected to the destabilization of medial meniscus (DMM) model of post-traumatic OA. The cKO mice showed reduced cartilage degradation and collagenase activity and fewer hypertrophy-like features at 12 weeks after DMM. Interestingly, in spite of the protection from structural articular cartilage damage, the postnatal growth plates of IKKα cKO mice after DMM displayed abnormal architecture and composition associated with increased chondrocyte apoptosis, which were not as evident in the articular chondrocytes of the same animals. Together, our results provide evidence of a novel in vivo functional role for IKKα in cartilage degradation in post-traumatic OA, and also suggest intrinsic, cell-autonomous effects of IKKα in chondrocytes that control chondrocyte phenotype and impact on cell survival, matrix homeostasis, and remodeling.

Elf3 Contributes to Cartilage Degradation in vivo in a Surgical Model of Post-Traumatic Osteoarthritis.

The E-74 like factor 3 (ELF3) is a transcription factor induced by inflammatory factors in various cell types, including chondrocytes. ELF3 levels are elevated in human cartilage from patients with osteoarthritis (OA), and ELF3 contributes to the IL-1ß-induced expression of genes encoding Mmp13, Nos2, and Ptgs2/Cox2 in chondrocytes in vitro. Here, we investigated the contribution of ELF3 to cartilage degradation in vivo, using a mouse model of OA. To this end, we generated mouse strains with cartilage-specific Elf3 knockout (Col2Cre:Elf3f/f) and Comp-driven Tet-off-inducible Elf3 overexpression (TRE-Elf3:Comp-tTA). To evaluate the contribution of ELF3 to OA, we induced OA in 12-week-old Col2Cre:Elf3f/f and 6-month-old TRE-Elf3:Comp-tTA male mice using the destabilization of the medial meniscus (DMM) model. The chondrocyte-specific deletion of Elf3 led to decreased levels of IL-1ß- and DMM-induced Mmp13 and Nos2 mRNA in vitro and in vivo, respectively. Histological grading showed attenuation of cartilage loss in Elf3 knockout mice compared to wild type (WT) littermates at 8 and 12 weeks following DMM surgery that correlated with reduced collagenase activity. Accordingly, Elf3 overexpression led to increased cartilage degradation post-surgery compared to WT counterparts. Our results provide evidence that ELF3 is a central contributing factor for cartilage degradation in post-traumatic OA in vivo.

Progressive cell-mediated changes in articular cartilage and bone in mice are initiated by a single session of controlled cyclic compressive loading.

We previously showed that repetitive cyclic loading of the mouse knee joint causes changes that recapitulate the features of osteoarthritis (OA) in humans. By applying a single loading session, we characterized the temporal progression of the structural and compositional changes in subchondral bone and articular cartilage. We applied loading during a single 5-minute session to the left tibia of adult (26-week-old) C57Bl/6 male mice at a peak load of 9.0N for 1,200 cycles. Knee joints were collected at times 0, 1, and 2 weeks after loading. The changes in articular cartilage and subchondral bone were analyzed by histology, immunohistochemistry (caspase-3 and cathepsin K), and microcomputed tomography. At time 0, no change was evident in chondrocyte viability or cartilage or subchondral bone integrity. However, cartilage pathology demonstrated by localized thinning and proteoglycan loss occurred at 1 and 2 weeks after the single session of loading. Transient cancellous bone loss was evident at 1 week, associated with increased osteoclast number. Bone loss was reversed to control levels at 2 weeks. We observed formation of fibrous and cartilaginous tissues at the joint margins at 1 and 2 weeks. Our findings demonstrate that a single session of noninvasive loading leads to the development of OA-like morphological and cellular alterations in articular cartilage and subchondral bone. The loss in subchondral trabecular bone mass and thickness returns to control levels at 2 weeks, whereas the cartilage thinning and proteoglycan loss persist. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1941-1949, 2016.

Mouse models of osteoarthritis: surgical model of posttraumatic osteoarthritis induced by destabilization of the medial meniscus.

The surgical model of destabilization of the medial meniscus (DMM) has become a gold standard for studying the onset and progression of posttraumatic osteoarthritis (OA). The DMM model mimics clinical meniscal injury, a known predisposing factor for the development of human OA, and permits the study of structural and biological changes over the course of the disease. In addition, when applied to genetically modified or engineered mouse models, this surgical procedure permits dissection of the relative contribution of a given gene to OA initiation and/or progression. This chapter describes the requirements for the surgical induction of OA in mouse models, and provides guidelines and tools for the subsequent histological, immunohistochemical, and molecular analyses. Methods for the assessment of the contributions of selected genes in genetically modified strains are also provided.

ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.

Endochondral ossification is a highly regulated process that relies on properly orchestrated cell-cell interactions in the developing growth plate. This study is focused on understanding the role of a crucial regulator of cell-cell interactions, the membrane-anchored metalloproteinase ADAM17, in endochondral ossification. ADAM17 releases growth factors, cytokines, and other membrane proteins from cells and is essential for epidermal growth factor receptor (EGFR) signaling and for processing tumor necrosis factor alpha. Here, we report that mice lacking ADAM17 in chondrocytes (A17ΔCh) have a significantly expanded zone of hypertrophic chondrocytes in the growth plate and retarded growth of long bones. This abnormality is caused by an accumulation of the most terminally differentiated type of chondrocytes that produces a calcified matrix. Inactivation of ADAM17 in osteoclasts or endothelial cells does not affect the zone of hypertrophic chondrocytes, suggesting that the main role of ADAM17 in the growth plate is in chondrocytes. This notion is further supported by in vitro experiments showing enhanced hypertrophic differentiation of primary chondrocytes lacking Adam17. The enlarged zone of hypertrophic chondrocytes in A17ΔCh mice resembles that described in mice with mutant EGFR signaling or lack of its ligand transforming growth factor α (TGFα), suggesting that ADAM17 regulates terminal differentiation of chondrocytes during endochondral ossification by activating the TGFα/EGFR signaling axis.