Kaneyuki Tsuchimochi

The control of chondrogenesis

Chondrogenesis is the earliest phase of skeletal development, involving mesenchymal cell recruitment and migration, condensation of progenitors, and chondrocyte differentiation, and maturation and resulting in the formation of cartilage and bone during endochondral ossification. This process is controlled exquisitely by cellular interactions with the surrounding matrix, growth and differentiation factors, and other environmental factors that initiate or suppress cellular signaling pathways and transcription of specific genes in a temporal‐spatial manner. Vertebrate limb development is controlled by interacting patterning systems involving prominently the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and hedgehog pathways. Both positive and negative signaling kinases and transcription factors, such as Sox9 and Runx2, and interactions among them determine whether the differentiated chondrocytes remain within cartilage elements in articular joints or undergo hypertrophic maturation prior to ossification. The latter process requires extracellular matrix remodeling and vascularization controlled by mechanisms that are not understood completely. Recent work has revealed novel roles for mediators such as GADD45β, transcription factors of the Dlx, bHLH, leucine zipper, and AP‐1 families, and the Wnt/β‐catenin pathway that interact at different stages during chondrogenesis. J. Cell. Biochem.

Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism

In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45β and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morphogenetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-κB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.

Cytoplasmic destruction of p53 by the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin'

Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum ‐associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin‐null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.

Differential expression of GADD45β in normal and osteoarthritic cartilage: Potential role in homeostasis of articular chondrocytes

OBJECTIVE Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage. METHODS Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin. RESULTS GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes. CONCLUSION These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

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-1β stimulation in chondrocytes, and the IL-1β-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-1β-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.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium‐specific ETS (ESE)‐1 transcription factor is induced in chondrocytes by interleukin‐1β (IL‐1β). We reported previously that early activation of EGR‐1 by IL‐1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE‐1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF‐κB‐dependent inhibition by IL‐1β. Of the ETS factors tested, this response was specific to ESE‐1, since ESE‐3, which was also induced by IL‐1β, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS‐1 increased COL2A1 promoter activity and blocked the inhibition by IL‐1β. These responses to ESE‐1 and ETS‐1 were confirmed using siRNA‐ESE1 and siRNA‐ETS1. In transient cotransfections, the inhibitory responses to ESE‐1 and IL‐1β colocalized in the −577/−132 bp promoter region, ESE‐1 bound specifically to tandem ETS sites at −403/−381 bp, and IL‐1‐induced binding of ESE‐1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE‐1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS‐1, and other IL‐1‐induced factors, including EGR‐1 at any given time. Intracellular ESE‐1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE‐1 in cartilage degeneration and suppression of repair. J. Cell. Physiol. 215: 562–573, 2008.

A cis-regulatory site downregulates PTHLH in translocation t(8;12)(q13;p11.2) and leads to Brachydactyly Type E

Parathyroid hormone-like hormone (PTHLH) is an important chondrogenic regulator; however, the gene has not been directly linked to human disease. We studied a family with autosomal-dominant Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation with breakpoints (BPs) upstream of PTHLH on chromosome 12p11.2 and a disrupted KCNB2 on 8q13. We sequenced the BPs and identified a highly conserved Activator protein 1 (AP-1) motif on 12p11.2, together with a C-ets-1 motif translocated from 8q13. AP-1 and C-ets-1 bound in vitro and in vivo at the derivative chromosome 8 breakpoint [der(8) BP], but were differently enriched between the wild-type and BP allele. We differentiated fibroblasts from BDE patients into chondrogenic cells and found that PTHLH and its targets, ADAMTS-7 and ADAMTS-12 were downregulated along with impaired chondrogenic differentiation. We next used human and murine chondrocytes and observed that the AP-1 motif stimulated, whereas der(8) BP or C-ets-1 decreased, PTHLH promoter activity. These results are the first to identify a cis-directed PTHLH downregulation as primary cause of human chondrodysplasia.

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

GADD45β (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 Gadd45β−/− 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 GADD45β 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 GADD45β together with AP-1, ATF, or C/EBP family members. C/EBPβ expression also predominantly co-localizes with GADD45β in the embryonic growth plate. Moreover, GADD45β enhances C/EBPβ activation via MTK1, MKK3, and MKK6, and dominant-negative p38αapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45β and C/EBPβ on the Col10a1 promoter. Importantly, GADD45β knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPβ binding to the Col10a1 promoter in vivo, indicating that GADD45β influences the transactivation function of DNA-bound C/EBPβ. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPβ is the target of the GADD45β-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45β via the MTK1/MKK3/6/p38 axis to C/EBPβ-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.

Identification of a Crucial Site for Synoviolin Expression

ABSTRACT Synoviolin is an E3 ubiquitin ligase localized in the endoplasmic reticulum (ER) and serving as ER-associated degradation system. Analysis of transgenic mice suggested that synoviolin gene dosage is implicated in the pathogenesis of arthropathy. Complete deficiency of synoviolin is fatal embryonically. Thus, alternation of Synoviolin could cause breakdown of ER homeostasis and consequently lead to disturbance of cellular homeostasis. Hence, the expression level of Synoviolin appears to be important for its biological role in cellular homeostasis under physiological and pathological conditions. To examine the control of protein level, we performed promoter analysis to determine transcriptional regulation. Here we characterize the role of synoviolin transcription in cellular homeostasis. The Ets binding site (EBS), termed EBS-1, from position −76 to −69 of the proximal promoter, is responsible for synoviolin expression in vivo and in vitro. Interestingly, transfer of EBS-1 decoy into NIH 3T3 cells conferred not only the repression of synoviolin gene expression but also a decrease in cell number. Fluorescence-activated cell sorter analysis using annexin V staining confirmed the induction of apoptosis by EBS-1 decoy and demonstrated recovery of apoptosis by overexpression of Synoviolin. Our results suggest that transcriptional regulation of synoviolin via EBS-1 plays an important role in cellular homeostasis. Our study provides novel insight into the transcriptional regulation for cellular homeostasis.

Senescence of chondrocytes in aging articular cartilage: GADD45β mediates p21 expression in association with C/EBPβ in senescence-accelerated mice

Growth arrest and DNA damage-inducible protein 45β (GADD45β) is expressed in normal and early osteoarthritic articular cartilage. We recently reported that GADD45β enhances CCAAT/enhancer binding protein β (C/EBPβ) activation in vitro. This study was undertaken in order to determine whether GADD45β is expressed with C/EBPβ in aging articular cartilage. We also investigated whether the synergistic expression of GADD45β and C/EBPβ may be involved in the mechanism of chondrocyte senescence. Senescence-accelerated mice (SAMP1) were used as a model of aging. GADD45β, C/EBPβ, and p21 were analyzed by immunohistochemistry. A luciferase reporter assay using ATDC5 cells was performed in order to examine p21 as a target gene of the GADD45β/C/EBPβ cascade. GADD45β exhibited increased expression in the aging articular cartilage of SAMP1 mice compared to that in control mice. The co-localization of GADD45β and C/EBPβ was confirmed by double immunostaining. The synergistic mechanisms of GADD45β and C/EBPβ on the gene regulation of p21, a molecule related to cellular senescence, were verified by a p21-luciferase reporter assay. Co-expression of C/EBPβ and p21 was confirmed. These observations suggest that the synergism between GADD45β and C/EBPβ may play an important role in cellular senescence in the aging articular cartilage.